Conjugates of a pharmaceutical agent and a moiety capable of binding to a glucose sensing protein

ABSTRACT

The invention describes novel conjugates of formula (I) of a pharmaceutical agent and a moiety capable of binding to a glucose sensing protein allowing a reversible release of the pharmaceutical agent depending on the glucose concentration.

The invention describes novel conjugates of a pharmaceutical agent and amoiety capable of binding to a glucose sensing protein allowing areversible release of the pharmaceutical agent depending on the glucoseconcentration.

Over the last decades the number of patients suffering from diseases,particulary from type 2 diabetes, has increased dramatically. Despiteeducation and treatment the growth rate is exploding. The diseaseevolves slowly and in the beginning the pancreas can compensatedecreasing insulin sensitivity by an increased release of insulin. Atthis stage oral antidiabetics like insulin sensitizers and -releaserscan support this compensation mechanism, but cannot cure the disease. Soafter this period of time external insulin has to be injected.

Several insulins are on the market, which are classified by theirduration of action. The intrinsic danger of hypoglycemia is counteractedby very flat insulin profiles (so called basal insulins), but is neitherconceptionally addressed nor finally overcome by these basal insulins.

The development of a real glucose sensing insulin accomplishing aglucose dependent release from a depot simulating the natural release bythe pancreas is still one of the holy grails in diabetes research. Suchan insulin would generate a local (eg intraparenteral) or moving depot(blood streem) from where it is released in a glucose concentrationdependent manner and finally recaptured by the system on decreasingglucose concentrations.

The blood glucose concentration is under hormonal regulation. Whileseveral hormones like glucagon, epinephrine, norepinephrine, cortisol,and hormones from the thyroid gland provoke elevated glucose levels,insulin is the only hormone which lowers glucose levels. In addition theglucose level is of course influenced by timing and composition ofmeals, physical stress, and infections.

In healthy persons the fasting blood glucose level is around 5 mM (900mg/L) and can after a meal increase to 40 mM for several hours. Indiabetic patients where blood glucose is out of control, the level canvary between 1-30 mM and can unpredictable fluctuate between the bordersof hyperglycemia (>10 mM) and hypoglycemia (<3 mM). Despite thepossibility of exact blood glucose measurement and titration ofinsulins, hypoglycemia is still a serious problem. This problem can besolved by glucose sensitive and -responsive delivery of pharmaceuticalagents effecting the glucose level.

Non glucose-sensitive depots to protect drugs (small molecules andproteins like insulin) from degradation and elongate their half life areused frequently in medicine. For insulin for example a staticsubcutanous depot can be realized. Insulin is stored as insolublehexamers. From this depot soluble monomers are released to the bloodfollowing law of mass equation.

An additional opportunity is the non-covalent binding of modifiedinsulins to albumin. Since unmodified insulin is not binding to albumin,noncovalent hydrophobic binding is enabeled by hydrophobic modification(eg by myristic acid). Coupling of fatty acids to insulin enableprotection of insulin from degradation and dramatically increases halflife by hours to days.

The release of insulin from such a circulating depot can be described bythe law of mass equation and is a function of the amount of insulin, thealbumin depot, and the affinity of the insulin derivative to albumin.Since the depot is fixed, the amount and affinity of insulin have to beadjusted. The release of basal insulin can be controlled, but therelease is glucose independent.

Within the last decade efforts have been started to establish glucosesensitive insulin depots. These efforts can be summarized and assignedto three classical principles:

-   -   Chemical recognition of glucose by boronic acids    -   Biochemical recognition of glucose by carbohydrate binding        proteins like such as lectins (Concanavalin A, wheat germ        agglutinin)    -   Glucose converting enzymes like glucose oxidase or hexokinase.        Here binding affinity can be used as a signal. More frequently        associated pH shift or change of charge is measured.

These principles can be used for glucose measurement or to translate thesignals into direct or indirect glucose release. Four possibilities forrealization are described below.

-   -   Direct modification of insulins    -   “Glucose responsive” hydrogels, these are synthetic pores, which        are modified with a glucose sensing molecule (boronic acid- or        glucose oxidase based). These gels are filled with insulins. In        the presence of glucose they expand, get leaky, and finally        release insulin on increasing glucose levels.    -   “Device-approaches”: In this case insulin levels are only        measured by a sensor.    -   Closed loop approaches: This describes a technical solution. A        sensor measures glucose levels. The signal is transmitted to an        independent insulin depot (eg a pump) which releases insulin        triggered by the signal. An independent insulin reservoir is        triggered and releases insulin, controlled by the sensor signal.        An advantage may be a large insulin depot which is not        necessarily in the body.

Several patent applications, e.g. WO 2001/92334, WO 2011/000823, or WO2003/048195 describe the use of boronic acid modified insulinderivatives in combination with albumin for a glucose sensitive insulinrelease. With this approach the floating insulin/albumin depot shall befurther developed to a glucose sensing floating depot.

A different approach for a glucose sensing approach has been describedin WO 2010/088294, WO 2010/88300, WO 2010/107520, WO 2012/015681, WO2012/015692, or WO 2015/051052. These documents describe the concomitantadministration of concanavalin A and a glucose binding proteinpreferably recognizing mannose. Accordingly mannose modified insulinscan be released by mannose from a depot. In addition an intrinsicmannose binding protein is described which may be responsible for thebinding of mannose without the need of concanavalin.

Erythrocytes have been used as a vehicle for the transport of drugs,e.g. for tumor starvation, enzyme replacement and immunotherapy asdescribed in WO 2015/121348, WO 2014/198788 and WO 2013/139906.

Liu et al. (Bioconjugate Chem. 1997, 8, 664-672) discloses a glucoseinduced release of glucosylpoly(ethylene glycol) insulin bound to asoluble conjugate of concanavalin A wherein the insulin is linked at theB1 amino group with a poly(ethylene glycol) spacer to the 1-position ofthe sugar.

WO2012/177701 discloses conjugates of ⁶⁸Ga-DOTA labelled sugars fortissue specific disease imaging and radiotherapy.

The use of erythrocytes as a classical depot, by binding drugs to thesurface of erythrocytes is described in WO 2013/121296. Here peptidesare described, which bind to the surface with a very high affinity(K_(D)=6.2 nM). These peptides are used for immunomodulation e.g. intransplantation medicine.

The present invention relates to a novel conjugate comprising apharmaceutical agent and a sugar moiety.

Further the present invention relates to a novel conjugate comprising apharmaceutical agent and a sugar moiety for use as a pharmaceutical.

Further the present invention relates to a novel conjugate comprising apharmaceutical agent and a sugar moiety which binds to the insulindependent glucose transporter GluT1, which provides a release of thepharmaceutical agent dependent on the glucose concentration in blood.The insulin dependent glucose transporter GluT1 is present onerythrocytes. Binding of glucose to GluT1 is reversible based on theblood glucose concentration.

In one embodiment the conjugate of the invention is bound to GluT1 atlow glucose concentrations of e.g. 1-10 mM, which are found underfasting conditions. Under these conditions, the stable floating depot ofthe active agent is formed. After an increase in the glucoseconcentration from e.g. 30 mM to 40 mM after a meal, the free glucosecompetes for the GluT1 binding site and the conjugate is released in aglucose concentration dependent manner and the pharmaceutical agent isavailable to exert its effect. As the glucose concentration decreasesagain, the conjugate molecules are recaptured by GluT1. Thus, thepresence of undesired high amounts of free pharmaceutical agents isavoided.

The present invention relates to conjugates of formula (I):

P-[L₁]_(m)-[A₁]_(o)-[L₂]_(p)-[A₂]_(r)-[L₃]_(q)-S   (I)

wherein P is a pharmaceutical agent, particularly a peptide,L₁, L₂, and L₃ are independently of each other a linker having a chainlength of 1-20 atoms,A₁ and A₂ are independently of each other a 5 to 6 membered monocyclicring or a 9 to 12 membered bicyclic ring, or two 5 to 6 memberedmonocyclic and/or 9 to 12 membered bicyclic rings connected to eachother, wherein each ring is independently a saturated, unsaturated, oraromatic carbocyclic or heterocyclic ring and wherein each ring maycarry at least one substituent,S is a sugar moiety which binds to the insulin independent glucosetransporter GluT1, andm, o, p, r, and q are independently of each other 0 or 1, and wherein atleast one of r and o is 1,or a pharmaceutically acceptable salt or solvate thereof.

The present invention relates also to conjugates of formula (I):

P-[L₁]_(m)-[A₁]_(o)-[L₂]_(p)-[A₂]_(r)-[L₃]_(q)-S   (I)

wherein P is an insulin or an insulinotropic peptide,L₁, L₂, and L₃ are independently of each other a linker having a chainlength of 1-20 atoms,A₁ and A₂ are independently of each other a 5 to 6 membered monocyclicring or a 9 to 12 membered bicyclic ring, or two 5 to 6 memberedmonocyclic and/or 9 to 12 membered bicyclic rings connected to eachother, wherein each ring is independently a saturated, unsaturated, oraromatic carbocyclic or heterocyclic ring and wherein each ring maycarry at least one substituent,S is a sugar moiety which binds to the insulin independent glucosetransporter GluT1, and comprises a terminal pyranose S1 moiety which isattached via position 2, 4, or 6 to the conjugate of formula (I),m, o, p, r, and q are independently of each other 0 or 1, and wherein atleast one of r and o is 1,or a pharmaceutically acceptable salt or solvate thereof.

Another aspect of the invention are compounds of formula (Ia) and (Ib):

R—(O═C)-[L₁]_(m)-[A₁]_(o)-[L₂]_(p)-[A₂]_(r)-[L₃]_(q)-S   (Ia)

[L₁]_(m)-[A₁]_(o)-[L₂]_(p)-[A₂]_(r)-[L₃]_(q)-S   (Ib)

wherein L₁, L₂, L₃, A₁, A₂, S, m, o, p, r, and q are defined asindicated above and R is H, halogen, OH, O-alkyl-, an anhydride forminggroup or another active ester forming group for coupling reactions, like4-nitrophenylester, succinate or N-hydroxy benzotriazol.or pharmaceutically acceptable salts or solvates thereof.

Compounds (Ia) and (Ib) are suitable as intermediates for the synthesisof the conjugates of formula (I).

Another aspect of the present invention is the conjugate of formula (I)as described above for the use in medicine, particularly in humanmedicine.

Another aspect of the present invention is a pharmaceutical compositioncomprising a conjugate of formula (I) as described above as an activeagent and a pharmaceutically acceptable carrier.

Another aspect of the present invention is a method of preventing and/ortreating a disorder associated with, caused by, and/or accompanied by adysregulated glucose metabolism, comprising administering a conjugate offormula (I) or a composition as described above to a subject in needthereof, particularly a human patient.

Another aspect of the present invention is a method of preventing and/ortreating diabetes type 1 or diabetes type 2.

The conjugates of formula (I) of the present invention comprise apharmaceutical agent P, which may a biomolecule, such as a peptide.Preferably, the pharmaceutical agent has an effect of directly orindirectly lowering the glucose concentration in blood. For example, thepharmaceutical agent may be an insulin or an insulinotropic peptide.

The term “insulin” according to the present invention encompasses humaninsulin, porcine insulin, or analogs thereof, e.g. prandial insulinswith fast action or basal insulins with long action. For example, theterm “insulin” encompasses recombinant human insulin, insulin glargine,insulin detemir, insulin glulisine, insulin aspart, insulin lispro, etc.or an insulin conjugated to a polyethylene, e.g. a low molecular weightPEG having a molecular weight of 10 kDa or less. If P is an insulin, itmay be attached via an amino group to form the conjugate of formula (I),e.g. via an amino side chain, particularly via the amino side chain ofan insulin B29Lys residue or via the amino terminus of an insulin B1Pheresidue.

Further, the pharmaceutical agent may be an insulinotropic peptide suchas GLP-1, an exendin such as exendin-4, or a GLP-1 agonist such aslixisenatide, liraglutide.

The conjugate of formula (I) further comprises a sugar moiety whichbinds to the insulin independent glucose transporter GluT1, also knownas solute carrier family 2, facilitated glucose transporter member 1(SLC2A1). The amino acid sequence of the human protein is NP_006507,which is encoded by a nucleic acid sequence NM_006516. GluT1 is anintegral membrane protein which facilitates diffusion of glucose intothe erythrocyte. The highest expression of GluT1 is found inerythrocytes.

For interaction with GluT1, the conjugate of formula (I) comprises amoiety binding to GluT1 but preventing transport through the erythrocytemembrane. A sugar moiety binding to GluT1 is preferably in an anomericform, particularly in an anomeric 6-membered ring form such as apyranose moiety. The sugar moiety comprises an anomeric O atom as wellas a hydroxy group or a protected hydroxy group at position 3 andposition 4 of a pyranose backbone. In one embodiment, the sugar moiety Sof the conjugate of formula (I) comprises a terminal pyranose moietywhich is attached via position 2, position 4, or position 6 of thepyranose backbone moiety.

Further, an aspect of the present invention is that introduction of atleast one cyclic residue A₁ and/or A₂ adjacent to the sugar moietycauses a substantial increase in the affinity to GluT1 in comparison toglucose.

Thus, the present invention provides a pharmaceutical agent in form of aconjugate of formula (I) which forms an erythrocyte-based circulatingdepot that after administration releases/delivers the agent as afunction of glucose concentration. Accordingly at low glucoseconcentrations (below 3 mM) no or only low concentration of free unboundlevels of the conjugate should be detectable. On increasing bloodglucose levels after a meal the conjugate is released from thecirculating depot into the blood stream. The release is a consequence ofa direct competition of glucose with the conjugate of formula (I). Thus,release is described by the law of mass equation und self adjusts totiniest changes in glucose levels. The same should be true for there-capturing process of the conjugate of formula (I) on decreasingglucose levels.

These characteristics constitute an essential advantage in comparison tothe glucose sensing depots from the prior art.

By means of the present invention, the drawbacks of prior art insulinswith regard to glycemia are diminished or avoided. The control ofglucose recognition and associated release/retrapping will be realizedwithin a single molecule. This minimizes delays in release/retrapping.Glucose sensitive binding and -release is controlled by interaction withendogenous transport and recognition processes. The biologicalrecognition system based on GluT1 transport in erythrocytes isconstantly regenerated by the organism.

The present conjugate of formula (I) binds to the ubiquitary glucosetransporter GluT1, which has a binding affinity to glucose in the samerange as glucose oxidase, a protein frequently used in glucoserecognition. GluT1 is highly expressed in erythrocytes and isresponsible for the basal supply of these cells. The size of the depotis large enough to accommodate the amount of pharmaceutical agent neededwithout affecting the erythrocyte glucose supply.

The affinity of the present conjugate of formula (I) is within anaffinity window which guarantees binding at low (e.g. <3 mM) glucoselevels. With increasing glucose levels (e.g. >10 mM) the conjugate offormula (I) is released accordingly. With decreasing glucose levels theunbound conjugate of formula (I) is recaptured by the transporter.

The release is following the law of mass equation and is dependent onthe size of the depot, the loading, and the affinity of the conjugate offormula (I) to GluT1. Since the depot is fixed, the free conjugatefraction is defined by the affinity to GluT1.

In certain embodiments, the conjugate of formula (I) has an affinity of10-500 nM to the insulin independent glucose transporter GluT1 asdetermined by affinity measurements for example by a ligand displacementassay, by MST (microscale thermophoresis) technology.

In the conjugate of formula (I) of the present invention, the individualstructural moieties P, A₁, A₂, and S may be connected by linkers L₁, L₂,and L₃. If present, L₁, L₂, and L₃ are linkers having a chain length of1-20 atoms, particularly 3 to 10, or 3 to 6 atoms.

In some embodiments, L₁, L₂, and L₃ are independently of each other(C₁-C₂₀) alkylene, (C₂-C₂₀) alkenylene, or (C₂-C₂₀) alkynylene, whereinone or more C-atoms may be replaced by heteroatoms or heteroatommoieties, particularly by O, NH, N(C₁₋₄) alkyl, S, SO₂, O—SO₂, O—SO₃,O—PHO₂ or O—PO₃, and/or wherein one or more C-atoms may be substitutedwith (C₁₋₄) alkyl, (C₁₋₄) alkyloxy, oxo, carboxyl, halogen, e.g. F, Cl,Br, or I, or a phosphorus-containing group. The carboxyl group may be afree carboxylic acid group or a carboxylic acid ester, e.g. C₁-C₄ alkylester or a carboxamide or mono(C₁-C₄) alkyl or di(C₁-C₄) alkylcarboxamide group. An example of a phosphorus-containing group is aphosphoric acid or phosphoric acid (C₁₋₄) alkyl ester group.

In certain embodiments, the linker L₃ has a chain length of 1 to 15atoms, particularly 1 to 6 or 1 to 4 atoms. For example, L₃ may be a(C₁-C₆) alkylene, particularly (C₁₋₄) alkylene group, wherein one or twoC-atoms may be replaced by heteroatoms or heteroatom moieties,particularly by O, NH, N(C₁₋₄) alkyl, S, SO₂, O—SO₂, O—SO₃, O—PHO₂ orO—PO₃, and/or wherein one or more C-atoms may be substituted with (C₁₋₄)alkyl, (C₁₋₄) alkyloxy, oxo, carboxyl, halogen, e.g. F, Cl, Br, or I, ora phosphorus-containing group.

In one embodiment, the linker L₃ is C═O.

In one embodiment, the linker L₃ is absent.

In one embodiment, the linker L₂ is —CO—(CH₂)₃—.

In one embodiment, the linker L₂ is —(CH₂)₆—NH—.

In one embodiment, the linker L₂ is —(CH₂)₂—CO—(CH₂—CH₂—O)₂—(CH₂)₂—NH—.

In one embodiment, the linker L₂ is —CH₂—O—(CH₂—CH₂—O)₃—.

The conjugate of formula (I) of the present invention comprises at leastone cyclic group, particularly a cyclic group A₂ and optionally afurther cyclic group A₁. An aspect of the present invention is that thepresence of a cyclic group adjacent to the sugar moiety S significantlyenhances the binding affinity of the sugar moiety S to the glucosetransporter GluT1. The cyclic groups A₁ and A₂ may be a 5 to 6 memberedmonocyclic ring, a 9 to 12 membered bicyclic ring, or two 5 to 6membered monocyclic and/or 9 to 12 membered bicyclic rings connected toeach other by a bond or 1-atom bridge, e.g. such as —O— or —CH₂—. Eachring may be a saturated, unsaturated, or aromatic carbocyclic orheterocyclic ring. Each ring may be unsubstituted or carry at least onesubstituent, for example, 1 to 3 substituents selected from halogen,NO₂, CN, (C₁₋₄) alkyl, (C₁₋₄) alkoxy, (C₁₋₄)alkyl-(C₃₋₇)cycloalkyl,(C₃₋₇) cycloalkyl, OH, benzyl, —O-benzyl, carboxyl, carboxyester,carboxamide, or mono (C₁₋₄) alkyl, or di (C₁₋₄) alkyl carboxamide.

In a further embodiment, A₂ and/or A₁ are a heterocyclic ring wherein 1to 4 ring atoms, e.g. 1, 2, 3, or 4 ring atoms are selected fromnitrogen, sulfur and/or oxygen and wherein the ring may be unsubstitutedor may carry at least one substituent as described above. In anespecially preferred embodiment, A₂ and A₁, if present, areindependently of each other a 5 to 6 membered monocyclic ring, whereinthe ring is a heteroalkyl ring, particularly selected from pyrrolidinyl,pyrazolidinyl, imidazolidinyl, thiazolidinyl, piperazinyl, piperidinyl,morpholinyl, wherein the ring may carry at least one substituent, or a 9to 12 membered bicyclic ring wherein the ring is a heteroalkyl ring with1 to 4 ring atoms being selected from N, O, and/or S, and wherein thering may carry at least one substituent.

In an especially preferred embodiment, A₂ and A₁, if present, areselected from pyrrolidinyl, pyrazolidinyl, imidazolidinyl,triazolidinyl, piperazinyl, piperidinyl, morpholinyl.

In a further embodiment A₂ and/or A₁ are 1,2,3-triazolyl.

In a further embodiment A₂ is 1,2,3-triazolyl.

In a further embodiment A₂ is piperazinyl.

A further group of embodiments are conjugates of formula (I) wherein A₂is piperazinyl, L₂ is absent and A₁ is cyclohexanyl.

A further group of embodiments are conjugates of formula (I) wherein A₂is piperazinyl, L₂ is absent and A₁ is cyclohexanyl.

A further group of embodiments are conjugates of formula (I) wherein A₂is piperazinyl, L₂ is —CH₂— and A₁ is cyclohexanyl.

A further group of embodiments are conjugates of formula (I) wherein A₂is piperazinyl, L₂ is absent and A₁ is phenyl.

A further group of embodiments are conjugates of formula (I) wherein A₂is 1,2,3-triazolyl, L₂ is absent and A₁ is phenyl.

A further group of embodiments are conjugates of formula (I) wherein

L₃ is —CO—, A₁ is phenyl, L₂ is —O— and A₁ is phenyl wherein each ringmay be unsubstituted or carry at least one substituent, for example, 1to 3 substituents selected from halogen, NO₂, CN, (C₁₋₄) alkyl, (C₁₋₄)alkoxy, (C₁₋₄)alkyl-(C₃₋₇)cycloalkyl, (C₃₋₇) cycloalkyl, OH, benzyl,—O-benzyl, carboxyl, carboxyester, carboxamide, or mono (C₁₋₄) alkyl, ordi (C₁₋₄) alkyl carboxamide.

A further group of embodiments are conjugates of formula (I) wherein thegroup -A₂-L₃- is selected from

In a further embodiment of the present invention, the conjugate offormula (I) comprises a single cyclic group A₂ and the second cyclicgroup A₁ is absent. In such embodiments, the conjugate of formula (I)may have a structure wherein m=1, o=0, p=0, and q=0 or 1. In otherembodiments, a second cyclic group A₁ is present. In such embodiments,the conjugates of formula (I) may have a structure wherein m=1, o=1,p=1, and q=0 or 1.

The conjugate of formula (I) comprises a sugar moiety S which binds tothe insulin independent glucose transporter GluT1. This sugar moiety Smay comprise a terminal pyranose moiety which is attached via position2, 4, or 6 to the conjugate of formula (I). In one embodiment theterminal pyranose moiety is attached via position 6 to the conjugate offormula (I).

In some embodiments, the sugar moiety S may comprise a terminal pyranosemoiety S1 having a backbone structure of Formula (II)

wherein 1, 2, 3, 4, 5, and 6 denote the positions of the C-atoms in thepyranose moiety,wherein

is a single bond and

is a single or a double bond, andR1 and R3 are H or a protecting group,and wherein S1 is attached via position 2, 4, or 6 to the conjugate offormula (I).

The protecting group may be any suitable protecting group known in theart, e.g. an acyl group such as acetyl or benzoyl, an alkyl group suchas methyl, an aralkyl group such as benzyl, or 4-methoxybenzyl (PMB)including divalent protecting groups such as isopropylidene orbenzylidene.

In some embodiments, the terminal pyranose moiety may be selected fromglucose, galactose, 4-deoxyglucose, and 4,5-dehydroglucose derivatives,wherein the terminal pyranose moiety is attached via position 2, 4, or 6to the conjugate of formula (I) or is mannose attached via position 6.

In another embodiment, the terminal pyranose moiety S1 is of the Formula(IIIa) or (IIIb):

wherein R1 is H or a protecting group such as methyl or acetyl,R2 is OR8, or NHR8 or an attachment site to the conjugate of formula(I), wherein R8 is H or a protecting group such as acetyl or benzyl,R3 is H or a protecting group such as acetyl or benzyl,R4 is H, OR8, or NHR8 or an attachment site to the conjugate of formula(I), wherein R8H or a protecting group such as acetyl or benzyl,or R1 and R2 and/or R3 and R4 form together with the pyranose ring atomsto which they are bound a cyclic group, e.g. an acetal,R5 and R6 are H or together form together with the carbon atom to whichthey are bound a carbonyl group,R7 is OR8, or NHR8 or an attachment site to the conjugate of formula(I), wherein R8 is H or a protecting group such as acetyl or benzyl, andwherein one of R2, R4, and R7 is the attachment site to the conjugate offormula (I).

In another embodiment of the terminal pyranose moiety S1 of the formula(IIIa) and (IIIb), R1 and R3 are H. In further embodiments of theterminal pyranose moiety S1 of the formula (IIIa) and (IIIb), R2 is OR8,or an attachment site to the conjugate of formula (I), R4 is H, OR8, oran attachment site to the conjugate of formula (I), R7 is OR8 or anattachment site to the conjugate of formula (I), and wherein R8 is H ora protecting group.

In another embodiment of the terminal pyranose moiety S1 of the formula(IIIa) and (IIIb), position 6 of the pyranose moiety and particularlysubstituent R7 is the attachment site of the terminal pyranose moiety S1to the conjugate of formula (I).

In specific embodiments, the pyranose moiety S1 is of formula (IVa),(IVb), (IVc), (IVd), or (IVe):

wherein R1, R2, R3, R5, R6, and R7 are defined as indicated above,wherein R4a is H or the attachment site to the conjugate of formula (I),and wherein R4 is H, a protecting group, or the attachment site to theconjugate of formula (I).

The sugar moiety S of the conjugate of formula (I) may comprise one ormore, e.g. 2, or 3 saccharide units. For example, the sugar moiety has astructure of formula (V):

—[X₂—S2]_(s)-X₁—S1   (V)

wherein X₁ is a bond or O, particularly a bond,X₂ is a bond, NH or O, particularly a bond,S2 is a mono- or disaccharide moiety, particularly comprising at leastone hexose or pentose moiety,S1 is a terminal pyranose moiety as defined above, ands is 0 or 1.

The saccharide moiety S2 may be a pyranose moiety, particularly selectedfrom glucose, galactose, 4-deoxyglucose, and 4,5-dehydroglucosederivatives or a furanose moiety, particularly selected from fructosederivates.

In specific embodiments, the saccharide moiety S2 is of formula (VIa),(VIb), (Vlc), (VId), or (VIe):

wherein R11 is a bond to X₁,R12 is OR8 or NHR8 or an attachment site to X₂, wherein R8 is H or aprotecting group such as acetyl or benzyl,R13 is H or a protecting group such as acetyl or benzyl,R14 is R8 or an attachment site to X₂, wherein R8 is H or a protectinggroup such as acetyl,R14a is H or an attachment site to X₂,R15 and R16 are H or together form together with the carbon atom towhich they are bound a carbonyl group,R17 is OR8 or an attachment site to X₂, wherein R8 is H or a protectinggroup such as acetyl or benzyl,or R11 and R12 and/or R13 and R14 form together with the ring atoms towhich they are bound a cyclic group such as an acetal,and wherein one of R12, R14, R14a and R17 is an attachment site to X₂.

In further embodiments, the conjugate of formula (I) reversibly binds tothe insulin independent glucose transporter GluT1, dependent from theglucose concentration in the surrounding medium, which is blood afteradministration. In a further embodiment the conjugate of formula (I) ofthe present invention is not transported through the cell membrane uponbinding to GluT1. In a further embodiment the sugar moiety S comprises asingle terminal saccharide moiety. In still further embodiments, thesugar moiety S does not comprise a mannose unit, particularly a terminalmannose unit.

Definitions

“Alkyl” means a straight-chain or branched carbon chain. Alkyl groupsmay be unsubstituted or substituted, wherein one or more hydrogens of analkyl carbon may be replaced by a substituent such as halogen. Examplesof alkyl include methyl, trifluoromethyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl.

“Alkylene” means a straight-chain or branched carbon chain bonded toeach side. Alkylene groups may be unsubstituted or substituted.

“Aryl” refers to any substituent derived from a monocyclic or polycyclicor fused aromatic ring, including heterocyclic rings, e.g. phenyl,thiophene, indolyl, naphthyl, pyridyl, which may optionally be furthersubstituted.

“Acyl” means a chemical functional group of the structure R—(C═O)—,wherein R is an alkyl, aryl, or aralkyl.

“Halogen” means fluoro, chloro, bromo, or iodo. Preferably, halogen isfluoro or chloro.

A “5 to 7 membered monocyclic ring” means a ring with 5 or 7 ring atomsthat may contain up to the maximum number of double bonds (aromatic ornon-aromatic ring which is fully, partially or un-saturated) wherein atleast one ring atom up to 4 ring atoms may be replaced by a heteroatomselected from the group consisting of sulfur (including —S(O)—,—S(O)₂—), oxygen and nitrogen (including ═N(O)—). Examples for 5 to 7membered rings include carbocycles such as cyclopentane, cyclohexane,and benzene, or heterocycles such as furan, thiophene, pyrrole,pyrroline, imidazole, imidazoline, pyrazole, triazole, pyrazoline,oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline,isothiazole, isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran,tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine,oxazolidine, isoxazolidine, thiazolidine, isothiazolidine,thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran,imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine,piperidine, morpholine, tetrazole, triazole, triazolidine,tetrazolidine, diazepame, azepine, or homopiperazine.

“9 to 12 membered bicyclic ring” means a system of two rings with 9 to12 ring atoms, where at least one ring atom is shared by both rings andthat may contain up to the maximum number of double bonds (aromatic ornon-aromatic ring which is fully, partially or un-saturated) wherein atleast one ring atom up to 6 ring atoms may be replaced by a heteroatomselected from the group consisting of sulfur (including —S(O)—,—S(O)₂—), oxygen, and nitrogen (including ═N(O)—) and wherein the ringis linked to the rest of the molecule via a carbon or nitrogen atom.Examples for 9 to 12 membered rings include carbocycles such asnaphthalene and heterocycles such as indole, indoline, benzofuran,benzothiophene, benzoxazole, benzisoxazole, benzothiazole,benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline,dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline,decahydroquinoline, isoquinoline, decahydroisoquinoline,tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine, orpteridine. The term 9 to 12 membered heterobicycle also includes spirostructures of two rings like 1,4-dioxa-8-azaspiro[4.5]decane or bridgedheterocycles like 8-aza-bicyclo[3.2.1]octane.

The term “protecting group” means a chemical protecting group forprotecting OH— groups, known in the art of sugar chemistry as describedin Theodora W. Greene, Peter G. M. Wuts, Protective Groups in OrganicSynthesis, 3rd Edition, John Wiley & Sonc, Inc. 1999. Examples of aprotecting group are: acetyl, benzyl, or p-methoxybenzyl; orisopropylidene groups for protecting two hydroxy groups.

The term “anhydride forming group” means a chemical group which formswith the carbonyl group to which it is attached an anhydride. An exampleis acetic anhydride which acetylates said carbonyl group.

The term “active ester forming group” means a chemical group which formswith the carbonyl group to which it is attached an ester which activatessaid carbonyl group for a coupling reaction with an amino groupcontaining conpound forming an amide group.

Examples of active ester forming groups are 4-Nitrophenylester,N-Hydroxybenzotriazol (HOBt), 1-Hydroxy-7-azabenzotriazol oderN-Hydroxysuccinimid (HOSu).

The term “pharmaceutically acceptable” means approved by a regulatoryagency such as the EMEA (Europe) and/or the FDA (US) and/or any othernational regulatory agency for use in animals, and/or in humans.

The conjugate of formula (I) of the present invention is suitable foruse in medicine, e.g. in veterinary medicine or in human medicine.Particularly, the conjugate of formula (I) is suitable for humanmedicine. Due to the glucose dependent release/recapture mechanism, theconjugate of formula (I) is particularly suitable for use in theprevention and/or treatment of disorders associated with, caused by,and/or accompanied by a dysregulated glucose mechanism, for example foruse in the prevention and/or treatment of diabetes mellitus,particularly of diabetes type 2 or type 1.

The invention also provides a pharmaceutical composition comprising aconjugate of formula (I) as described above as an active agent and apharmaceutically acceptable carrier.

The term “pharmaceutical composition” indicates a mixture containingingredients that are compatible when mixed and which may beadministered. A pharmaceutical composition includes one or moremedicinal drugs. Additionally, the pharmaceutical composition mayinclude one or more pharmaceutically acceptable carriers such assolvents, adjuvants, emollients, expanders, stabilizers, and othercomponents, whether these are considered active or inactive ingredients.

The conjugates of formula (I) of the present invention, or saltsthereof, are administered in conjunction with an acceptablepharmaceutical carrier as part of a pharmaceutical composition. A“pharmaceutically acceptable carrier” is a compound or mixture ofcompounds which is physiologically acceptable while retaining thetherapeutic properties of the substance with which it is administered.Standard acceptable pharmaceutical carriers and their formulations areknown to one skilled in the art and described, for example, inRemington: The Science and Practice of Pharmacy, (20th ed.) ed. A. R.Gennaro A. R., 2000, Lippencott Williams & Wilkins. One exemplarypharmaceutically acceptable carrier is physiological saline solution.

Acceptable pharmaceutical carriers include those used in formulationssuitable for oral, rectal, nasal, or parenteral (including subcutaneous,intramuscular, intravenous, intradermal, and transdermal)administration. The compounds of the present invention will typically beadministered parenterally.

The term “pharmaceutically acceptable salt” means salts of theconjugates of formula (I) of the invention which are safe and effectivefor use in mammals. Pharmaceutically acceptable salts may include, butare not limited to, acid addition salts and basic salts. Examples ofacid addition salts include chloride, sulfate, hydrogen sulfate,(hydrogen) phosphate, acetate, citrate, tosylate, or mesylate salts.Examples of basic salts include salts with inorganic cations, e.g.alkaline or alkaline earth metal salts such as sodium, potassium,magnesium, or calcium salts and salts with organic cations such as aminesalts. Further examples of pharmaceutically acceptable salts aredescribed in Remington: The Science and Practice of Pharmacy, (20th ed.)ed. A. R. Gennaro A. R., 2000, Lippencott Williams & Wilkins or inHandbook of Pharmaceutical Salts, Properties, Selection and Use, e.d. P.H. Stahl, C. G. Wermuth, 2002, jointly published by Verlag HelveticaChimica Acta, Zurich, Switzerland, and Wiley-VCH, Weinheim, Germany.

The term “solvate” means complexes of the conjugates of formula (I) ofthe invention or salts thereof with solvent molecules, e.g. organicsolvent molecules and/or water.

The compounds of the present invention will be administered in a“therapeutically effective amount”. This term refers to a nontoxic butsufficient amount of the conjugate of formula (I) to provide the desiredeffect. The amount of a conjugate of formula (I) of the formula (I)necessary to achieve the desired biological effect depends on a numberof factors, for example the specific conjugate of formula (I) chosen,the intended use, the mode of administration, and the clinical conditionof the patient. An appropriate “effective” amount in any individual casemay be determined by one of ordinary skill in the art using routineexperimentation.

Pharmaceutical compositions of the invention are those suitable forparenteral (for example subcutaneous, intramuscular, intradermal, orintravenous), oral, rectal, topical, and peroral (for examplesublingual) administration, although the most suitable mode ofadministration depends in each individual case on the nature andseverity of the condition to be treated and on the nature of theconjugate of formula (I)) used in each case.

Suitable pharmaceutical compositions may be in the form of separateunits, for example capsules, tablets, and powders in vials or ampoules,each of which contains a defined amount of the conjugate of formula (I);as powders or granules; as solution or suspension in an aqueous ornonaqueous liquid; or as an oil-in-water or water-in-oil emulsion. Itmay be provided in single dose injectable form, for example in the formof a pen. The compositions may, as already mentioned, be prepared by anysuitable pharmaceutical method which includes a step in which the activeingredient and the carrier (which may consist of one or more additionalingredients) are brought into contact.

The conjugates of formula (I) of the present invention can be widelycombined with other pharmacologically active compounds, such as alldrugs mentioned in the Rote Liste 2016 e.g. with all antidiabeticsmentioned in the Rote Liste 2016, chapter 12.

The active ingredient combinations can be used especially for asynergistic improvement in action. They can be applied either byseparate administration of the active ingredients to the patient or inthe form of combination products in which a plurality of activeingredients are present in one pharmaceutical preparation. When theactive ingredients are administered by separate administration of theactive ingredients, this can be done simultaneously or successively.

General methods for the synthesis of conjugates of formula (I) andintermediates thereof are described in the following schemes:

OH-protected glucuronic acid compounds 4, where the protecting groups(PG) are e.g. acetyl, benzyl, or p-methoxybenzyl, or isopropylidenegroups for protecting two hydroxy groups at the same time, or the like,can be coupled with amines 5 using well known amide coupling procedurese.g. using HATU, TBTU, BEP, TOTU, or other activating methods forcarboxylic acids in common known solvents like dimethylformamide,tetrahydrofuran, dichlormethane, acetonitrile, or the like. Dependent onthe protecting group, the deprotection to compounds 2aa takes placeunder different conditions such as basic, acidic, hydrogenating oroxidative conditions. For example, acetyl groups are cleaved under basicconditions using sodium or lithium hydroxide in solvents like methanol,water, tetrahydrofuran, or combinations thereof. Isopropylidene groupsare cleaved under acidic conditions, e.g. using trifluoroacetic acid inwater, hydrogenating conditions using e.g. palladium on charcoal orother hydrogenating catalysts under hydrogen atmosphere in solvents likemethanol, ethanol, toluene, acetic acid, tetrahydrofuran or the like, oroxidative conditions like cerium ammonium nitrate or DDQ, like forp-methoxybenzyl.

Unsaturated compounds of formula 2ba can be synthesized like shown inscheme 2:

Starting from acetyl protected glucuronic acid, deacetylation usingacetic acid anhydride and triethylamine generates compound 7, which canbe coupled with compounds 5 using coupling reagents for amide bondsyntheses, like described above, to give compounds 2ba.

A further method to synthesize compounds 2ba is shown in scheme 3:

Starting from 1,2,3,4-tetra acetyl protected glucose, oxidation usingSwern conditions leads to aldehyde 8. Reductive amination of aldehyde 8and amines 5 using reductive amination conditions like sodiumcyanoborohydride, sodium triacetoxyborohydride or the like, in solventslike dichloroethane, dichloromethane, methanol, and/or acetic acid leadsto compounds 2ba.

Compounds 10, 11, and 12 can be synthesized as described in scheme 4.

Compounds of formula 10, 11, and 12 can be synthesized using coppercatalysed [3+2]-cycloaddition conditions, also known as azide-alkyne orclick cycloaddition. 1-, 2- or 6-azido-deoxyglucose and alkynes 9, arereacted with CuSO₄*5H₂O, tris(3-hydroxypropyltriazolylmethyl)amine(THPTA) and sodium ascorbate.

Alkynes 9 can be synthesized as shown in scheme 5.

Alkynes 9 can be synthesized using propargylamine under differentreaction conditions: alkylation conditions using bases likediisopropylamine, triethylamine or the like, in presence of differentchlorides or halides like for 9a and 9c, or peptide coupling conditionslike for 9b.

Compounds 14, 17, and 18 can be synthesized as described in scheme 6.

Compounds 14 can be synthesized using known deprotection methods. Whenprotecting group PG is, for example, a benzyl group, it can bedeprotected under hydrogenation conditions as described above. Alkynes15 can be converted into alkylating reagents 16 with R describing aleaving group like O-tosyl, O-mesyl, halogen or the like. Compounds 14and compounds 16 can be reacted to obtain compounds 17 under alkylatingconditions, e.g. using bases like triethylamine, diisopropylamine,sodium hydride or the like in aprotic solvents like dimethylformamide,tetrahydrofuran, toluene or the like. Deprotection of compounds 17 tocompounds 18 is dependent of the used protecting groups, the conditionsare as described above.

Galactosyl derivatives 20 can be synthesized like described in scheme 7.

Reductive amination of isopropylidene protected galactosyl-aldehyde andamines 5 using known reductive amination conditions as described inscheme 3 lead to compounds 19. Deprotection of the isopropylidene groupscan be done as described in scheme 1.

The synthesis of compounds 1 is described in scheme 8.

The synthesis of compounds 22 can be done under reductive aminationconditions for compounds 21 and protected carbohydrate aldehydes asdescribed in scheme 3. Deprotection of compounds 22 to compounds 23 canbe done as described in scheme 1. Compounds 23 can be coupled usingcopper-catalyzed azide-alkyne cycloaddition conditions as described inscheme 4 to yield compounds 1.

The synthesis of compounds 24 is described in scheme 9.

The synthesis of compounds 24 can be carried out by reaction ofcompounds 25 with insulin under basic conditions, e.g. pH 10. Thereforethe insulin is dissolved in a dimethylformamide-water mixture andbrought to pH 10 by an organic base like triethylamine. At lowtemperatures (e.g. 0° C.) the activated azido-dioxopyrrolidines 25 areadded to yield compounds of formula 24.

Abbreviations

-   BEP 2-bromo-1-ethyl pyridinium tetrafluoroborate-   d Dublet-   dd Double dublet-   ddd Double double dublet-   DDQ 2,3-dichloro-5,6-dicyano-1,4-benzochinone-   DMSO dimethylsulfoxide-   ELSD Evaporative Light Scattering Detector-   Eq. Equivalent/s-   ES-API Electro spray atmospheric pressure ionisation-   FCS Fetal calf serum-   HATU    1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-   3-oxide hexafluorophosphate-   HOBt 1-hydroxybenzotriazole-   HPLC High pressure liquid chromatography-   Hz Hertz-   J coupling constant-   KRB Krebs-Ringer bicarbonate buffer-   LG leaving group-   LC/MS Liquid chromatography/mass spectra-   m multiplet-   MEM Minimum-Essential-Medium-   MHz Megahertz-   MPLC Medium pressure liquid chromatography-   NEAA Non-essential amino acids-   NMR Nuclear magnetic resonance-   PG Protecting group-   q quadruplet-   s singulet-   t triplet-   td dublet of triplets-   TBTU N,N,N′,N′-Tetramethyl-O-(benzotriazol-1-yl)uronium    tetrafluoroborate-   TLC Thin layer chromatography-   THPTA tris(3-hydroxypropyltriazolylmethyl)amine-   TOTU    O-[(ethoxycarbonyl)cyanomethylenamino]-N,N,N′,N′-tetramethyluronium    tetrafluoroborate-   t_(R) Retention time-   R_(f) Relative to front value-   UV Ultra violet-   v/v Volume by volume

EXPERIMENTAL PART Chromatographic and Spectroscopic Methods TLC/UV-Lamp

Thin layer chromatography (TLC) was done on glass plates from Merckcoated with silica gel 60 F254. Detection was done with an UV-Lamp fromLamag at wavelengths of 254 nm and 366 nm.

Compounds which could not be detected by UV were stained by differentmethods: (a) 10% H₂SO₄ in ethanol, b) 1% KMnO₄-solution, c)molybdatophosphoric acid-cerium(IV)sulfate solution in sulfuric acid (6mL concentrated sulfuric acid and 94 mL water, 2.5 g molybdatophosphoricacid, 1 g cerium(IV)sulfate).

MPLC

Chromatography on normal phase was done on a CombiFlash® Rf (TeledyneISCO). The used gradients were given in the description of the examples.

HPLC

Preparative reversed phase HPLC was done using acetonitrile/water on anAgilent 1200 preparative HPLC machine and an Agilent Prep-C₁₈ column (10μm, 21.5×150 mm).

¹H-NMR

For ¹H-NMR-spectra a Bruker ARX, 400 MHz device was used.

¹³C-NMR

For ¹³C-NMR-spectra a Bruker Avance, 600 MHz device was used.

LC/MS

For retention time and mass detection a LC/MS-system from Waters AcquitySDS with a Waters Acquity BEH C₁₈ (1.7 μm, 2.1×50 mm) column was used.The injection volume was 0.5 μl. Molecular weights are given in grammper mol [g/mol], detected masses in mass per charge [m/e].

LC/MS—Method 1

95% H₂O (0.05% formic acid) to 95% acetonitrile (0.035% formic acid) in2 min, 95% acetonitrile till 2.60 min, 0.9 mL/min, 10×2 mm PhenomenexLunaC₁₈ 3 μm.

LC/MS—Method 2

93% H₂O (0.05% trifluoroacetic acid) to 95% acetonitrile (0.05%trifluoroacetic acid) in 1 min, 95% acetonitrile till 1.45 min, 1.1mL/min, 10×2.0 mm LunaC₁₈ 3 μm

LC/MS—Method 3

99% H₂O (0.05% trifluoroacetic acid) to 93% H₂O (0.05%) in 0.4 min, 95%acetonitrile (0.05% trifluoroacetic acid) in 0.8 min, 95% acetonitriletill 1.8 min, 1.1 mL/min, 10×2.0 mm LunaC₁₈ 3 μm

LC/MS—Method 4

10% acetonitrile (0.1% formic acid) till 90% acetonitrile (0.1% formicacid) in 10 min, 90% acetonitrile till 10.67 min, 10% acetonitrile from11 to 12 min, 0.5 mL/min, Aeris Widepore 3, 3 μm, 100×2.1 mm, 40° C.

Syntheses Method A Amide coupling with1,2,3,4-tetra-O-acetyl-β-D-glucuronic acid

To a solution of 0.55 mmol 1,2,3,4-tetra-O-acetyl-β-D-glucuronic acid in5 mL dimethylformamide were added 1.4 eq. (0.77 mmol)1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (HATU) and 1.4 eq. (0.77 mmol) amine. Thereaction mixture was stirred for 2-6 hours at room temperature. Reactioncontrol was done by TLC. As work up 5-10 mL dichloromethane were addedand the organic phase was washed with 1M HCl, water, saturated aqueousNaHCO₃ solution, and water. The organic phases were dried with Na₂SO₄,filtered, and evaporated. If needed the crude mixture was purified byMPLC.

Example 1[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-methylpiperazine-1-carbonyl)tetrahydropyran-4-yl]acetate

Example 1 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-methylpiperazine following the procedure described insynthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO): column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 254 nm; eluent:(A) dichloromethane, (B) ethanol.

Mplc Gradient:

start % B end % B duration [min] 0 0 2.2 0 4.8 2.8 4.8 4.8 6.0 4.8 10.16.0 10.1 10.1 9.1

Yield: 137 mg (0.308 mmol, 55.8%), white solid.

TLC: R_(f)=0.250 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=445.13 [M+H]⁺; calculated: 445.44; t_(R) (λ=220 nm):0.93 min (LC/MS—method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=5.96 (d, J=8.3 Hz, 1H, CH), 5.37 (t, J=9.5Hz, 1H, CH), 5.25 (t, J=9.5 Hz, 1H, CH), 4.97 (m, 2H, 2×CH), 3.63 (m,1H, NCH₂), 3.55 (m, 1H, CH₂), 3.39 (m, 1H, NCH₂), 3.23 (m, 1H, NCH₂),2.36 (m, 2H, NCH₂), 2.16 (s, 3H, CH₃), 2.11 (m, 2H, NCH₂), 2.08 (s, 3H,CH₃), 2.02 (s, 3H, CH₃), 1.93 (s, 3H, CH₃), 1.90 (s, 3H, CH₃) ppm.

Example 2[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-ethylpiperazine-1-carbonyl)tetrahydropyran-4-yl]acetate

Example 2 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-ethylpiperazine following the procedure described insynthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 2.2 0 4.8 2.8 4.8 4.8 6.0 4.8 10.16.0 10.1 10.1 9.1

Yield: 201 mg (0.438 mmol, 79.4%), white solid.

TLC: R_(f)=0.492 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=459.12 [M+H]⁺; calculated: 459.46; t_(R) (λ=220 nm):0.98 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=5.97 (d, J=8.3 Hz, 1H, CH), 5.38 (t, J=9.5Hz, 1H, CH), 5.25 (t, J=9.5 Hz, 1H, CH), 4.97 (m, 2H, 2×CH), 3.58 (m,2H, NCH₂), 3.40 (m, 1H, NCH₂), =3.24 (m, 1H, NCH₂), 2.41 (m, 2H, NCH₂),2.32 (m, 2H, CH₂), 2.16 (m, 2H, NCH₂), 2.08 (s, 3H, CH₃), 2.02 (s, 3H,CH₃), 1.93 (s, 3H, CH₃), 1.89 (s, 3H, CH₃), 0.99 (t, J=7.1 Hz, 3H, CH₃)ppm.

Example 3[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-n-propylpiperazine-1-carbonyl)tetrahydropyran-4-yl]acetate (8)

Example 3 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-n-propylpiperazine following the procedure described insynthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B Duration [min] 0 0 1.5 0 5.0 5.0 5.0 5.0 8.0

Yield: 144 mg (0.305 mmol, 55.2%), white solid.

TLC: R_(f)=0.417 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=473.14 [M+H]⁺; calculated: 473.89; t_(R) (λ=220 nm):1.06 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=5.96 (d, J=8.3 Hz, 1H, CH), 5.38 (t, J=9.5Hz, 1H, CH), 5.25 (t, J=9.5 Hz, 1H, CH), 4.98 (m, 2H, 2×CH), 3.58 (m,2H, NCH₂), 3.40 (m, 1H, NCH₂), 3.24 (m, 1H, NCH₂), 2.39 (m, 2H, NCH₂),2.22 (m, 2H, NCH₂), 2.15 (m, 2H, NCH₂), 2.07 (s, 3H, CH₃), 2.02 (s, 3H,CH₃), 1.93 (s, 3H, CH₃), 1.89 (s, 3H, CH₃), 1.43 (m, 2H, CH₂), 0.85 (t,J=7.3 Hz, 3H, CH₃) ppm.

Example 4[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-n-butylpiperazine-1-carbonyl)tetrahydropyran-4-yl]acetate

Example 4 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-n-butylpiperazine following the procedure described insynthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 1.0 0 10.1 20.0 10.1 10.1 3 10.10.0 0.0 0.0 0.0 1.0

Yield: 191 mg (0.393 mmol, 71.1%), white solid.

TLC: R_(f)=0.458 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=487.19 [M+H]⁺; calculated: 487.51; t_(R) (λ=220 nm):1.14 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=5.96 (d, J=8.4 Hz, 1H, CH), 5.38 (t, J=9.4Hz, 1H, CH), 5.25 (t, J=9.4 Hz, 1H, CH), 4.97 (m, 2H, 2×CH), 3.57 (m,2H, NCH₂), 3.40 (m, 1H, NCH₂), 3.24 (m, 1H, NCH₂), 2.40 (m, 2H, NCH₂),2.26 (m, 2H, NCH₂), 2.15 (m, 2H, NCH₂), 2.07 (s, 3H, CH₃), 2.02 (s, 3H,CH₃), 1.93 (s, 3H, CH₃), 1.90 (s, 3H, CH₃), 1.39 (m, 2H, CH₂), 1.28 (m,2H, CH₂), 0.87 (t, J=7.4 Hz, 3H, CH₃) ppm.

Example 5[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-n-hexylpiperazine-1-carbonyl)tetrahydropyran-4-yl]acetate

Example 5 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-n-hexylpiperazine following the procedure described insynthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 1.0 0 20.2 20.0 20.2 20.2 2.9 20.20.0 0.0 0.0 0.0 1.0

Yield: 226 mg (0.439 mmol, 79.6%), white solid.

TLC: R_(f)=0.489 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=515.24 [M+H]⁺; calculated: 515.57; t_(R) (λ=220 nm):1.34 min (LC/MS—Method 1).

¹H-NMR (400 MHz, 26.9° C., DMSO-d₆): δ=5.96 (d, J=8.3 Hz, 1H, CH), 5.37(t, J=9.5 Hz, 1H, CH), 5.25 (t, J=9.5 Hz, 1H, CH), 4.97 (m, 2H, CH),3.57 (m, 2H, NCH₂), 3.40 (m, 1H, CH₂), 3.23 (m, 1H, NCH₂), 2.39 (m, 2H,NCH₂), 2.25 (t, J=7.1 Hz, 2H, NCH₂), 2.15 (m, 2H, NCH₂), 2.16 (s, 3H,CH₃), 2.07 (s, 3H, CH₃), 2.02 (s, 3H, CH₃), 1.93 (s, 3H, CH₃), 1.89 (s,3H, CH₃), 1.40 (m, 2H, CH₂), 1.26 (m, 6H, 3×CH₂), 0.86 (t, J=6.9 Hz, 2H,CH₃) ppm.

Example 6[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-isopropylpiperazine-1-carbonyl)tetrahydropyran-4-yl]acetate

Example 6 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-isopropylpiperazine following the procedure described insynthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 1.0 0 20.2 20.0 20.2 20.2 2.9 20.20.0 0.0 0.0 0.0 1.0

Yield: 129 mg (0.273 mmol, 49.5%), white solid.

TLC: R_(f)=0.412 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=473.18 [M+H]⁺; calculated: 473.47; t_(R) (λ=220 nm):1.02 min (LC/MS—Method 1).

¹H-NMR (400 MHz, 26.9° C., DMSO-d₆): δ=5.97 (d, J=8.3 Hz, 1H, CH), 5.38(t, J=9.4 Hz, 1H, CH), 5.25 (t, J=9.4 Hz, 1H, CH), 4.97 (m, 2H, 2×CH),3.56 (m, 2H, NCH₂), 3.39 (m, 1H, CH₂), 3.24 (m, 1H, NCH₂), 2.66 (m, 1H,CH), 2.43 (m, 2H, NCH₂), 2.27 (m, 2H, NCH₂), 2.08 (s, 3H, CH₃), 2.02 (s,3H, CH₃), 1.93 (s, 3H, CH₃), 1.90 (s, 3H, CH₃), 0.95 (dd, 6H, CH₃) ppm.

Example 7[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-tert-butylpiperazine-1-carbonyl)tetrahydropyran-4-yl]acetate

Example 7 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-tert-butylpiperazine following the procedure described insynthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 1.0 0 20.2 20.0 20.2 20.2 2.9 20.20.0 0.0 0.0 0.0 1.0

Yield: 139 mg (0.286 mmol, 51.8%), white solid.

TLC: R_(f)=0.464 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=487.20 [M+H]⁺; calculated: 487.51; t_(R): 1.06 min(LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=5.97 (d, J=8.3 Hz, 1H, CH), 5.38 (t, J=9.5Hz, 1H, CH), 5.25 (t, J=9.5 Hz, 1H, CH), 4.97 (m, 2H, CH), 3.53 (m, 2H,NCH₂), 3.39 (m, 1H, CH₂), 3.26 (m, 1H, NCH₂), 2.67 (m, 2H, NCH₂), 2.32(m, 2H, NCH₂), 2.07 (s, 3H, CH₃), 2.02 (s, 3H, CH₃), 1.93 (s, 3H, CH₃),1.90 (s, 3H, CH₃), 0.99 (s, 9H, 3×CH₃) ppm.

Example 8[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-allylpiperazine-1-carbonyl)tetrahydropyran-4-yl]acetate

Example 8 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-allylpiperazine following the procedure described insynthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 1.0 0 9.9 9.9

Yield: 184 mg (0.260 mmol, 70.8%), white solid.

TLC: R_(f)=0.479 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=471.26 [M+H]⁺; calculated: 471.19; t_(R): 1.02 min(LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=5.97 (d, J=8.1 Hz, 1H, CH), 5.81 (m, 1H,H₂C═CH), 5.36 (t, J=9.4 Hz, 1H, CH), 5.24 (t, J=9.4 Hz, 1H, CH), 5.17(m, 2H, HC═CH₂), 4.98 (m, 2H, CH), 3.61 (m, 2H, NCH₂), 3.42 (m, 1H,NCH₂), 3.23 (m, 1H, NCH₂), 2.96 (m, 2H, NCH₂), 2.41 (m, 2H, NCH₂), 2.17(m, 2H, NCH₂), 2.08 (s, 3H, CH₃), 2.02 (s, 3H, CH₃), 1.94 (s, 3H, CH₃),1.90 (s, 3H, CH₃) ppm.

Example 9[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-cyclohexylpiperazine-1-carbonyl)tetrahydropyran-4-yl]acetate

Example 9 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-cyclohexylpiperazine following the procedure described insynthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 1.0 0 9.9 9.9

Yield: 227 mg (0.443 mmol, 80.2%), white solid.

TLC: R_(f)=0.610 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=513.15 [M+H]⁺; calculated: 513.24; t_(R): 1.22 min(LC/MS—Method 1).

¹H-NMR (400 MHz, 116.9° C., DMSO-d₆): 5.93 (d, J=7.8 Hz, 1H, CH), 5.33(m, 2H, 2×CH), 4.99 (m, 1H, CH), 4.88 (m, 1H, CH), 3.71 (m, 4H, NCH₂),3.04 (m, 6H, NCH₂), 3.23 (m, 2H, NCH₂), 2.36 (m, 2H, NCH₂), 2.06 (s, 3H,CH₃), 1.99 (s, 3H, CH₃), 1.93 (s, 3H, CH₃), 1.90 (s, 3H, CH₃), 1.82 (m,2H, CH₂), 1.63 (m, 1H, CH), 1.30 (m, 8H, CH₂) ppm.

Example 10[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(cyclohexylmethyl)piperazine-1-carbonyl]tetrahydropyran-4-yl]acetate

Example 10 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-cyclohexylmethylpiperazine following the procedure describedin synthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 1.0 0 20.0 20.0 20.0 20.0 3 20.00.0 0.0 0.0 0.0 1.0

Yield: 178 mg (0.338 mmol, 61.2%), white solid.

TLC: R_(f)=0.346 (ethylacetate/n-heptan, 2:1).

LC/MS (ES-API): m/z=527.22 [M+H]⁺; calculated: 527.25; t_(R): 1.31 min(LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=5.97 (d, J=8.3 Hz, 1H, CH), 5.39 (t, J=9.4Hz, 1H, CH), 5.27 (t, J=9.4 Hz, 1H, CH), 4.96 (m, 2H, CH), 3.58 (m, 2H,NCH₂), 3.40 (m, 1H, CH₂), 3.25 (m, 1H, NCH₂), 2.37 (m, 2H, NCH₂), 2.13(m, 2H, NCH₂), 2.19 (m, 2H, NCH₂), 2.08 (s, 3H, CH₃), 2.02 (s, 3H, CH₃),1.94 (s, 3H, CH₃), 1.89 (s, 3H, CH₃), 1.69 (m, 4H, 2×CH₂), 1.47 (m, 1H,CH), 1.18 (m, 2H, 2×CH₂), 0.82 (m, 2H, CH₂) ppm.

Example 11[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(2-cyclohexylethyl)piperazine-1-carbonyl]tetrahydropyran-4-yl]acetate

Example 11 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-cyclohexylethylpiperazine following the procedure describedin synthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 1.0 0 18.7 18.7

Yield: 183 mg (0.339 mmol, 61.3%), white solid.

TLC: R_(f)=0.511 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=541.24 [M+H]⁺; calculated: 541.27; t_(R): 1.41 min(LC/MS—Method 1).

¹H-NMR (400 MHz, 116.9° C., DMSO-d₆): δ=5.93 (d, J=7.8 Hz, 1H, CH), 5.33(m, 2H, 2×CH), 4.99 (m, 1H, CH), 4.88 (m, 1H, CH), 3.71 (m, 4H, NCH₂),3.04 (m, 6H, NCH₂), 3.23 (m, 2H, NCH₂), 2.36 (m, 2H, NCH₂), 2.06 (s, 3H,CH₃), 1.99 (s, 3H, CH₃), 1.93 (s, 3H, CH₃), 1.90 (s, 3H, CH₃), 1.71 (m,1H, CH), 1.61 (m, 6H, CH₂), 1.24 (m, 4H, CH₂), 1.02 (m, 2H, CH₂) ppm.

Example 12[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-phenylpiperazine-1-carbonyl)tetrahydropyran-4-yl]acetate

Example 12 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-phenylpiperazine following the procedure described insynthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 254 nm; eluent:(A) n-heptane, (B) ethylacetate.

Yield: 260 mg (0.514 mmol, 62.0%), white solid.

TLC: R_(f)=0.695 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=507.14 [M+H]⁺; calculated: 507.19; t_(R): 1.70 min(LC/MS—Method 1).

MPLC Gradient

start % B end % B duration [min] 0 0 1.3 0 100.0 13.7 100.0 100.0 3.7100.0 0.0 0.0 0.0 0.0 1.3

¹H-NMR (400 MHz, DMSO-d₆): δ=7.24 (t, J=8.2 Hz, 2H, ArH), 6.95 (d, J=8.2Hz, 2H, ArH), 6.81 (t, J=7.4 Hz, 1H, ArH), 6.00 (d, J=8.4 Hz, 1H, CH),5.40 (t, J=9.9 Hz, 1H, CH), 5.29 (t, J=9.9 Hz, 1H, CH), 5.05 (d, J=9.6Hz, 1H, CH), 5.00 (m, 1H, CH), 3.81 (m, 1H, NCH₂), 3.75 (m, 1H, NCH₂),3.57 (m, 1H, CH₂), 3.39 (m, 1H, NCH₂), 3.24 (m, 2H, NCH₂), 2.82 (m, 2H,NCH₂), 2.07 (s, 3H, CH₃), 2.03 (s, 3H, CH₃), 1.94 (s, 3H, CH₃), 1.91 (s,3H, CH₃) ppm.

¹³C-NMR (150 MHz, DMSO-d₆): δ=169.64 (s, C), 169.04 (s, C), 169.02 (s,C), 168.43 (s, C), 163.04 (s, C), 150.69 (s, C), 128.99 (s, CH), 119.47(s, CH), 115.97 (s, CH), 90.94 (s, CH), 72.02 (s, CH), 69.37 (s, CH),69.15 (s, CH), 68.55 (s, CH), 49.14 (s, CH₂), 48.07 (s, CH₂), 44.78 (s,CH₂), 41.41 (s, CH₃), 20.44 (s, 2 CH₃), 20.30 (s, 2 CH₃) ppm.

Example 13[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-[(E)-cinnamyl]piperazine-1-carbonyl]tetrahydropyran-4-yl]acetate

Example 13 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and trans-1-cinnamylpiperazine following the procedure described insynthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 1.0 0 9.9 9.9

Yield: 253 mg (0.463 mmol, 83.8%), white solid.

TLC: R_(f)=0.644 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=547.27 [M+H]⁺; calculated: 547.22; t_(R): 1.34 min(LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.44 (d, J=7.2 Hz, 2H, ArH), 7.32 (t, J=7.2Hz, 2H, ArH), 7.23 (t, J=7.2 Hz, 2H, ArH), 6.54 (d, J=15.9 Hz, 1H, CH),6.29 (m, 1H, CH), 5.96 (d, J=8.4 Hz, 1H, CH), 5.37 (t, J=9.5 Hz, 1H,CH), 5.25 (t, J=9.5 Hz, 1H, CH), 4.97 (m, 2H, CH), 3.61 (m, 2H, NCH₂),3.42 (m, 1H, CH₂), 3.26 (m, 1H, NCH₂), 3.12 (m, 2H, NCH₂), 2.47 (m, 2H,NCH₂), 2.22 (m, 2H, NCH₂), 2.06 (s, 3H, CH₃), 2.02 (s, 3H, CH₃), 1.93(s, 3H, CH₃), 1.89 (s, 3H, CH₃) ppm.

Example 14[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(4-chlorophenyl)piperazine-1-carbonyl]tetrahydropyran-4-yl]acetate

Example 14 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-(4-chlorophenyl)-piperazine following the procedure describedin synthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 2.4 0 5.0 7.9 5.0 5.0 3.7

Yield: 153 mg (0.282 mmol, 51.3%), white solid.

TLC: R_(f)=0.619 (ethylacetate/n-heptane, 2:1).

LC/MS (ES-API): m/z=541.19 [M+H]⁺; calculated: 541.15; t_(R): 1.80 min(LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.25 (d, J=9.0 Hz, 2H, ArH), 6.96 (d, J=9.0Hz, 2H, ArH), 6.00 (d, J=8.3 Hz, 1H, CH), 5.40 (t, J=9.5 Hz, 1H, CH),5.28 (t, J=9.5 Hz, 1H, CH), 5.01 (m, 2H, 2×CH), 3.76 (m, 2H, NCH₂), 3.56(m, 1H, NCH₂), 3.37 (m, 1H, NCH₂), 3.25 (m, 2H, NCH₂), 2.92 (m, 2H,NCH₂), 2.07 (s, 3H, CH₃), 2.03 (s, 3H, CH₃), 1.94 (s, 3H, CH₃), 1.90 (s,3H, CH₃) ppm.

Example 15[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(2-chlorophenyl)piperazine-1-carbonyl]tetrahydropyran-4-yl]acetate

Example 15 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-(2-chlorophenyl)-piperazine following the procedure describedin synthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 254 nm; eluent:(A) n-heptane, (B) ethylacetate.

MPLC Gradient

start % B end % B duration [min] 0 0 1.3 0 100.0 13.7 100.0 100.0 3.7100.0 0.0 0.0 0.0 0.0 1.3

Yield: 148 mg (0.274 mmol, 49.6%), white solid.

TLC: R_(f)=0.589 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=541.10 [M+H]⁺; calculated: 541.15; t_(R): 1.80 min(LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.44 (dd, J=1.4 Hz, 2H, ArH), 7.33 (m, 2H,ArH), 7.14 (dd, J=1.4 Hz, 2H, ArH), 7.07 (m, 2H, ArH), 5.98 (d, J=8.3Hz, 1H, CH), 5.39 (t, J=9.4 Hz, 1H, CH), 5.29 (t, J=9.4 Hz, 1H, CH),5.01 (m, 2H, 2×CH), 3.84 (m, 1H, NCH₂), 3.76 (m, 1H, NCH₂), 3.57 (m, 1H,CH₂), 3.38 (m, 1H, NCH₂), 3.02 (m, 2H, NCH₂), 2.80 (m, 2H, NCH₂), 2.08(s, 3H, CH₃), 2.03 (s, 3H, CH₃), 1.94 (s, 3H, CH₃), 1.92 (s, 3H, CH₃)ppm.

Example 16[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(4-bromophenyl)piperazine-1-carbonyl]tetrahydropyran-4-yl]acetate

Example 16 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-(4-bromophenyl)-piperazine following the procedure describedin synthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 1.0 0 20.2 20.0 20.2 20.2 2.9 20.20.0 0.0 0.0 0.0 1.0

Yield: 168 mg (0.287 mmol, 52.0%), white solid.

TLC: R_(f)=0.635 (ethylacetate/n-heptane, 2:1).

LC/MS (ES-API): m/z=585.14 [M+H]⁺; calculated: 585.10; t_(R): 1.82 min(LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.37 (d, J=9.0 Hz, 2H, ArH), 6.92 (d, J=9.0Hz, 2H, ArH), 6.00 (d, J=8.3 Hz, 1H, CH), 5.40 (t, J=9.4 Hz, 1H, CH),5.28 (t, J=9.4 Hz, 1H, CH), 5.01 (m, 2H, 2×CH), 3.76 (m, 2H, NCH₂), 3.56(m, 1H, NCH₂), 3.37 (m, 1H, NCH₂), 3.25 (m, 2H, NCH₂), 2.93 (m, 2H,NCH₂), 2.07 (s, 3H, CH₃), 2.03 (s, 3H, CH₃), 1.94 (s, 3H, CH₃), 1.90 (s,3H, CH₃) ppm.

Example 17[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(4-methoxyphenyl)piperazine-1-carbonyl]tetrahydropyran-4-yl]acetate

Example 17 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-(4-methoxyphenyl)-piperazine following the proceduredescribed in synthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 254 nm; eluent:(A) n-heptane, (B) ethylacetate.

MPLC Gradient

start % B end % B duration [min] 0 0 1.0 0 10.0 20.0 10.0 10.0 3.0 10.00.0 0.0 0.0 0.0 1.0

Yield: 192 mg (0.320 mmol, 58.0%), white solid.

TLC: R_(f)=0.508 (ethylacetate/n-heptane, 2:1).

LC/MS (ES-API): m/z=537.14 [M+H]⁺; calculated: 537.20; t_(R): 1.64 min(LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.91 (d, J=9.1 Hz, 2H, ArH), 6.83 (d, J=9.1Hz, 2H, ArH), 5.99 (d, J=8.0 Hz, 1H, CH), 5.40 (t, J=9.9 Hz, 1H, CH),5.28 (t, J=9.9 Hz, 1H, CH), 5.00 (m, 2H, 2×CH), 3.77 (m, 2H, NCH₂), 3.69(s, 3H, OCH₃), 3.56 (m, 1H, CH₂), 3.37 (m, 1H, NCH₂), 3.08 (m, 2H,NCH₂), 2.80 (m, 2H, NCH₂), 2.07 (s, 3H, CH₃), 2.03 (s, 3H, CH₃), 1.94(s, 3H, CH₃), 1.90 (s, 3H, CH₃) ppm.

Example 18[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(3-methoxyphenyl)piperazine-1-carbonyl]tetrahydropyran-4-yl]acetate

Example 18 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-(3-methoxyphenyl)-piperazine following the proceduredescribed in synthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 254 nm; eluent:(A) n-heptane, (B) ethylacetate.

MPLC Gradient

start % B end % B duration [min] 0 0 1.0 0 10.0 20.0 10.0 10.0 3.0 10.00.0 0.0 0.0 0.0 1.0

Yield: 120 mg (0.224 mmol, 40.6%), white solid.

TLC: R_(f)=0.571 (ethylacetate/n-heptane, 2:1).

LC/MS (ES-API): m/z=537.12 [M+H]⁺; calculated: 537.20; t_(R): 1.70 min(LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.12 (d, J=8.2 Hz, 1H, ArH), 6.83 (d, J=8.2Hz, 1H, ArH), 6.47 (s, 1H, ArH), 6.41 (d, J=8.2 Hz, 1H, ArH), 6.00 (d,J=8.0 Hz, 1H, CH), 5.39 (t, J=9.9 Hz, 1H, CH), 5.28 (t, J=9.9 Hz, 1H,CH), 5.00 (m, 2H, 2×CH), 3.78 (m, 2H, NCH₂), 3.72 (s, 3H, OCH₃), 3.56(m, 1H, CH₂), 3.37 (m, 1H, NCH₂), 3.24 (m, 2H, NCH₂), 2.92 (m, 2H,NCH₂), 2.08 (s, 3H, CH₃), 2.02 (s, 3H, CH₃), 1.96 (s, 3H, CH₃), 1.90 (s,3H, CH₃) ppm.

Example 19[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(2-methoxyphenyl)piperazine-1-carbonyl]tetrahydropyran-4-yl]acetate

Example 19 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-(2-methoxyphenyl)-piperazine following the proceduredescribed in synthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 2.5 0 5.0 8.5 5.0 5.0 3.9

Yield: 150 mg (0.280 mmol, 50.6%), white solid.

TLC: R_(f)=0.492 (ethylacetate/n-heptane, 2:1).

LC/MS (ES-API): m/z=537.14 [M+H]⁺; calculated: 537.20; t_(R): 1.67 min(LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.98 (t, J=4.1 Hz, 1H, ArH), 6.96 (t, J=4.1Hz, 1H, ArH), 6.89 (d, J=2.2 Hz, 2H, ArH), 6.86 (d, J=2.2 Hz, 2H, ArH),5.98 (d, J=8.4 Hz, 1H, CH), 5.40 (t, J=9.5 Hz, 1H, CH), 5.29 (t, J=9.5Hz, 1H, CH), 5.00 (m, 2H, 2×CH), 3.83 (m, 1H, NCH₂), 3.79 (s, 3H, OCH₃),3.74 (m, 1H, NCH₂), 3.70 (m, 1H, CH₂), 3.54 (m, 1H, NCH₂), 3.03 (m, 2H,NCH₂), 2.74 (m, 2H, NCH₂), 2.07 (s, 3H, CH₃), 2.03 (s, 3H, CH₃), 1.94(s, 3H, CH₃), 1.91 (s, 3H, CH₃) ppm.

Example 20[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(1,3-benzodioxol-5-ylmethyl)piperazine-1-carbonyl]tetrahydropyran-4-yl]acetate

Example 20 was synthesized from 1,2,3,4-tetra-O-acetyl-β-D-glucuronicacid and 1-(1,3-benzodioxol-5-ylmethyl)piperazine following theprocedure described in synthesis method A.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 1.0 0 10.0 20.0 10.0 10.0 3.0 10.00.0 0.0 0.0 0.0 1.0

Yield: 221 mg (0.391 mmol, 70.9%), white solid.

TLC: R_(f)=0.478 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=565.14 [M+H]⁺; calculated: 565.19; t_(R): 1.24 min(LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.84 (m, 2H, ArH), 6.73 (dd, J=1.4 Hz, 1H,ArH), 5.99 (s, 2H, O—CH₂—O), 5.95 (d, J=8.4 Hz, 1H, CH), 5.36 (t, J=9.5Hz, 1H, CH), 5.24 (t, J=9.5 Hz, 1H, CH), 4.96 (m, 2H, 2×CH), 3.62 (m,2H, NCH₂), 3.55 (m, 1H, CH₂), 3.39 (m, 1H, NCH₂), 3.23 (m, 1H, NCH₂),2.39 (m, 2H, NCH₂), 2.21 (m, 2H, NCH₂), 2.07 (s, 3H, CH₃), 2.02 (s, 3H,CH₃), 1.93 (s, 3H, CH₃), 1.88 (s, 3H, CH₃) ppm.

Method B

Deacetylation of Glucuronic Acid Amides

20 mg of[(2,3,5-triacetoxy-6-piperazine-1-carbonyl]tetrahydropyran-4-yl]acetates were dissolved in 2 mL methanol/H₂O/tetrahydrofuran (5:4:1) andcooled to 0° C. 20 μl of a 2 M lithium hydroxide solution in water wereadded and stirred for 2-12 hours at 0° C. The reaction control was doneby TLC and LC/MS. As work-up procedure the reaction mixture wasneutralized with 1M HCl, and the organic solvents were evaporated. Theresidue was diluted with water and lyophilized. The enantiomers were notseparated. NMR signals were listed for only one enantiomer.

Example 21(4-Methylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 21 was synthesized from example 1 following the deacetylationprocedure described in synthesis method B.

Yield: 11.9 mg (43.07 μmol, 95.7%), colorless oil.

LC/MS (ES-API): m/z=277.15 [M+H]⁺; calculated: 277.13; t_(R) (ELSD):0.21 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.51 (d, J=4.5 Hz, 1H, CH), 5.0-4.3 (m, 4H,4×OH), 3.5-3.0 (m, 12H, 4×CH₂, 4×CH), 2.21 (m, 3H, CH₃) ppm.

Example 22(4-Ethylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 22 was synthesized from example 2 following the deacetylationprocedure described in synthesis method B.

Yield: 11.8 mg (40.65 μmol, 93.2%), colorless oil.

LC/MS (ES-API): m/z=291.23 [M+H]⁺; calculated: 291.15; t_(R) (ELSD):0.20 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.48 (d, J=4.6 Hz, 1H, CH), 4.8-4.3 (m, 4H,4×OH), 3.5-3.0 (m, 12H, 4×CH₂, 4×CH), 2.20 (m, 5H, CH₂CH₃) ppm.

Example 23(4-n-Propylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 23 was synthesized from example 3 following the deacetylationprocedure described in synthesis method B.

Yield: 12.4 mg (40.74 μmol, 96.3%), colorless oil.

LC/MS (ES-API): m/z=305.21 [M+H]⁺; calculated: 305.16; t_(R) (ELSD):0.19 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.51 (d, J=4.4 Hz, 1H, CH), 4.9-4.1 (m, 4H,4×OH), 3.6-3.0 (m, 12H, 4×CH₂, 4×CH), 2.32 (m, 2H, NCH₂), 1.49 (m, 2H,CH₂), 0.89 (t, J=7.3 Hz, 3H, CH₃) ppm.

Example 24(4-n-Butylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 24 was synthesized from example 4 following the deacetylationprocedure described in synthesis method B.

Yield: 12.7 mg (39.89 μmol, 97.0%), colorless oil.

LC/MS (ES-API): m/z=319.21 [M+H]⁺; calculated: 319.18; t_(R) (ELSD):0.23 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.52 (d, J=4.5 Hz, 1H, CH), 4.87 (dd, J=4.2Hz, 1H, OH), 4.75 (dd, J=4.9 Hz, 1H, OH), 4.29 (d, J=8.9 Hz, 1H, OH),4.02 (d, J=9.4 Hz, 1H, OH), 3.6-3.15 (m, 12H, 4×CH₂, 4×CH), 2.32 (m, 2H,NCH₂), 1.46 (m, 2H, CH₂), 1.29 (m, 2H, CH₂), 0.89 (t, J=7.1 Hz, 3H, CH₃)ppm.

Example 25(4-n-Hexylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 25 was synthesized from example 5 following the deacetylationprocedure described in synthesis method B.

Yield: 12.6 mg (36.37 μmol, 93.6%), white solid.

LC/MS (ES-API): m/z=347.17 [M+H]⁺; calculated: 347.21; t_(R)1 (λ=220nm): 0.50 min; t_(R)2 (λ=220 nm): 0.53 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.50 (d, J=4.4 Hz, 1H, CH), 4.85 (dd, J=4.4Hz, 1H, OH), 4.76 (dd, J=4.8 Hz, 1H, OH), 4.27 (d, J=8.8 Hz, 1H, OH),4.12 (d, J=9.3 Hz, 1H, OH), 3.6-3.1 (m, 12H, 4×CH₂, 4×CH), 2.32 (m, 2H,NCH₂), 1.43 (m, 8H, 4×CH₂), 0.90 (t, J=7.2 Hz, 3H, CH₃) ppm.

Example 26(4-Isopropylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 26 was synthesized from example 6 following the deacetylationprocedure described in synthesis method B.

Yield: 12.2 mg (40.09 μmol, 94.7%), colorless oil.

LC/MS (ES-API): m/z=305.23 [M+H]⁺; calculated: 305.16; t_(R) (ELSD):0.21 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.48 (d, J=4.6 Hz, 1H, CH), 4.8-4.1 (m, 4H,4×OH), 3.5-3.1 (m, 12H, 4×CH₂, 4×CH), 2.43 (m, 2H, NCH), 1.01 (m, 6H,2×CH₃) ppm.

Example 27(4-tert-Butylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 27 was synthesized from example 7 following the deacetylationprocedure described in synthesis method B.

Yield: 12.5 mg (39.26 μmol, 95.5%), white solid.

LC/MS (ES-API): m/z=319.25 [M+H]⁺; calculated: 319.18; t_(R) (ELSD):0.22 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.49 (d, J=4.9 Hz, 1H, CH), 4.9-4.0 (m, 4H,4×OH), 3.5-3.1 (m, 12H, 4×CH₂, 4×CH), 1.04 (m, 9H, 3×CH₃) ppm.

Example 28(4-Allylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 28 was synthesized from example 8 following the deacetylationprocedure described in synthesis method B.

Yield: 12.4 mg (41.02 μmol, 96.5%), colorless oil.

LC/MS (ES-API): m/z=303.22 [M+H]⁺; calculated: 303.15; t_(R) (ELSD):0.21 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.29 (d, J=4.5 Hz, 1H, CH), 4.7-4.0 (m, 4H,4×OH), 3.4-3.1 (m, 12H, 4×CH₂, 4×CH), 2.41 (m, 5H, 2×CH₂, CH) ppm.

Example 29(4-Cyclohexylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 29 was synthesized from example 9 following the deacetylationprocedure described in synthesis method B.

Yield: 12.9 mg (37.46 μmol, 96.0%), white solid.

LC/MS (ES-API): m/z=345.25 [M+H]⁺; calculated: 345.19; t_(R)1 (ELSD):0.28 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.51 (d, J=4.8 Hz, 1H, CH), 4.6-4.0 (m, 4H,4×OH), 3.4-2.9 (m, 12H, 4×CH₂, 4×CH), 1.71 (m, 4H, 2×CH₂), 1.54 (m, 1H,CH), 1.19 (m, 6H, 3×CH₂) ppm.

Example 30(4-Cyclohexylmethylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 30 was synthesized from example 10 following the deacetylationprocedure described in synthesis method B.

Yield: 13.1 mg (36.55 μmol, 96.2%), white solid.

LC/MS (ES-API): m/z=359.16 [M+H]⁺; calculated: 359.21; t_(R)1 (λ=220nm): 0.46 min; t_(R)2 (λ=220 nm): 0.48 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.47 (d, J=4.5 Hz, 1H, CH), 4.9-4.3 (m, 4H,4×OH), 3.5-2.9 (m, 12H, 4×CH₂, 4×CH), 2.07 (m, 2H, NCH₂), 1.74 (m, 4H,2×CH₂), 1.52 (m, 1H, CH), 1.21 (m, 4H, 2×CH₂), 0.87 (m, 2H, CH₂) ppm.

Example 31(4-Cyclohexylethylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 31 was synthesized from example 11 following the deacetylationprocedure described in synthesis method B.

Yield: 12.2 mg (32.76 μmol, 88.5%), white solid.

LC/MS (ES-API): m/z=373.17 [M+H]⁺; calculated: 373.23; t_(R)1 (λ=220nm): 0.81 min; t_(R)2 (λ=220 nm): 0.84 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.42 (d, J=4.8 Hz, 1H, CH), 4.6-4.1 (m, 4H,4×OH), 3.6-3.0 (m, 12H, 4×CH₂, 4×CH), 2.05 (m, 2H, NCH₂), 1.72 (m, 4H,2×CH₂), 1.50 (m, 1H, CH), 1.23 (m, 4H, 2×CH₂), 0.90 (m, 2H, CH₂), 0.87(m, 2H, CH₂) ppm.

Example 32(4-Phenylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 32 was synthesized from example 12 following the deacetylationprocedure described in synthesis method B.

Yield: 11.8 mg (34.87 μmol, 88.3%), white solid.

LC/MS (ES-API): m/z=339.13 [M+H]⁺; calculated: 339.15; t_(R)1 (λ=220nm): 0.90 min; t_(R)2 (λ=220 nm): 0.95 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.23 (t, J=7.9 Hz, 2H, ArH), 6.95 (d, J=7.8Hz, 2H, ArH), 6.81 (t, J=7.3 Hz, 1H, ArH), 6.66 (d, J=7.0 Hz, 1H, OH),4.81 (d, J=4.7 Hz, 1H, OH), 4.74 (d, J=5.3 Hz, 1H, OH), 4.62 (d, J=6.4Hz, 1H, OH), 4.43 (t, J=7.3 Hz, 1H, CH), 4.09 (d, J=9.3 Hz, 1H, CH),3.70-3.45 (m, 8H, 4×NCH₂), 3.25-3.00 (m, 3H, 3×CH) ppm.

¹³C-NMR (150 MHz, DMSO-d₆): δ=166.48 (s, CO), 150.76 (s, C), 129.06 (s,CH), 119.28 (s, CH), 115.78 (s, CH), 93.06 (s, CH), 74.52 (s, CH), 72.71(s, CH), 71.33 (s, CH), 70.98 (s, CH), 48.93 (s, CH₂), 48.22 (s, CH₂),44.59 (s, CH₂), 41.15 (s, CH₂) ppm.

Example 33[4-[(E)-Cinnamyl]piperazin-1-yl]-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 33 was synthesized from example 13 following the deacetylationprocedure described in synthesis method B.

Yield: 12.7 mg (33.56 μmol, 91.7%), white solid.

LC/MS (ES-API): m/z=379.25 [M+H]⁺; calculated: 379.19; t_(R)1 (λ=220nm): 0.56 min; t_(R)2 (λ=220 nm): 0.60 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.40 (d, J=7.0 Hz, 2H, ArH), 7.33 (t, J=7.0Hz, 2H, ArH), 7.21 (t, J=7.0 Hz, 2H, ArH), 6.44 (d, J=4.6 Hz, 1H, CH),4.6-4.1 (m, 4H, 4×OH), 3.65 (m, 2H, NCH₂), 3.51 (m, 2H, 2×CH), 3.4-2.9(m, 12H, 4×NCH₂, 4×CH) ppm.

Example 34[4-(4-Chlorophenyl)piperazin-1-yl]-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 34 was synthesized from example 14 following the deacetylationprocedure described in synthesis method B.

Yield: 13.6 mg (36.48 μmol, 98.7%), white solid.

LC/MS (ES-API): m/z=373.16 [M+H]⁺; calculated: 373.11; t_(R)1 (λ=220nm): 1.20 min; t_(R)2 (λ=220 nm): 1.24 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.40 (d, J=7.8 Hz, 4H, ArH), 6.49 (d, J=4.7Hz, 1H, CH), 4.9-4.3 (m, 4H, 4×OH), 3.7-3.0 (m, 12H, 4×NCH₂, 4×CH) ppm.

Example 35[4-(2-Chlorophenyl)piperazin-1-yl]-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 35 was synthesized from example 15 following the deacetylationprocedure described in synthesis method B.

Yield: 13.3 mg (35.68 μmol, 96.5%), white solid.

LC/MS (ES-API): m/z=373.06 [M+H]⁺; calculated: 373.11; t_(R)1 (λ=220nm): 1.23 min; t_(R)2 (λ=220 nm): 1.26 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.45 (d, J=7.8 Hz, 1H, ArH), 7.31 (t, J=7.8Hz, 1H, ArH), 7.14 (d, J=7.8 Hz, 1H, ArH), 7.03 (t, J=7.8 Hz, 1H, ArH),6.52 (d, J=4.7 Hz, 1H, CH), 4.9-4.3 (m, 4H, 4×OH), 3.7-3.0 (m, 12H,4×NCH₂, 4×CH) ppm.

Example 36[4-(4-Bromophenyl)piperazin-1-yl]-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 36 was synthesized from example 16 following the deacetylationprocedure described in synthesis method B.

Yield: 12.9 mg (30.92 μmol, 90.5%), white solid.

LC/MS (ES-API): m/z=416.11 [M+H]⁺; calculated: 416.06; t_(R)1 (λ=220nm): 1.25 min; t_(R)2 (λ=220 nm): 1.28 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.69 (d, J=8.0 Hz, 4H, ArH), 6.52 (d, J=4.9Hz, 1H, CH), 4.8-4.1 (m, 4H, 4×OH), 3.6-3.1 (m, 12H, 4×NCH₂, 4×CH) ppm.

Example 37[4-(4-Methoxyphenyl)piperazin-1-yl]-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 37 was synthesized from example 17 following the deacetylationprocedure described in synthesis method B.

Yield: 13.4 mg (36.38 μmol, 97.6%), white solid.

LC/MS (ES-API): m/z=369.11 [M+H]⁺; calculated: 369.16; t_(R)1 (λ=220nm): 0.74 min; t_(R)2 (λ=220 nm): 0.80 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.91 (d, J=8.2 Hz, 2H, ArH), 6.83 (d, J=8.2Hz, 2H, ArH), 6.53 (d, J=4.4 Hz, 1H, CH), 4.8-4.1 (m, 4H, 4×OH), 3.69(s, 3H, OCH₃), 3.6-2.9 (m, 12H, 4×NCH₂, 4×CH) ppm.

Example 38[4-(3-Methoxyphenyl)piperazin-1-yl]-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 38 was synthesized from example 18 following the deacetylationprocedure described in synthesis method B.

Yield: 13.1 mg (35.56 μmol, 95.4%), white solid.

LC/MS (ES-API): m/z=369.10 [M+H]⁺; calculated: 369.16; t_(R)1 (λ=220nm): 1.01 min; t_(R)2 (λ=220 nm): 1.04 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.14 (d, J=8.2 Hz, 1H, ArH), 6.84 (d, J=8.2Hz, 1H, ArH), 6.57 (s, 1H, ArH), 6.51 (d, J=8.2 Hz, 1H, ArH), 6.43 (d,J=4.4 Hz, 1H, CH), 4.8-4.1 (m, 4H, 4×OH), 3.72 (s, 3H, OCH₃), 3.60-3.45(m, 4H, 4×CH), 3.25-3.00 (m, 8H, 4×NCH₂) ppm.

Example 39[4-(2-Methoxyphenyl)piperazin-1-yl]-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 39 was synthesized from example 19 following the deacetylationprocedure described in synthesis method B.

Yield: 12.8 mg (34.75 μmol, 93.2%), white solid.

LC/MS (ES-API): m/z=369.19 [M+H]⁺; calculated: 369.16; t_(R)1 (λ=220nm): 0.85 min; t_(R)2 (λ=220 nm): 0.90 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.98 (m, 2H, ArH), 6.87 (d, J=7.2 Hz, 2H,ArH), 6.49 (d, J=4.4 Hz, 1H, CH), 4.8-4.1 (m, 4H, 4×OH), 3.70 (s, 3H,OCH₃), 3.6-3.2 (m, 4H, 4×CH), 3.0-2.8 (m, 8H, 4×NCH₂) ppm.

Example 40[4-(1,3-Benzodioxol-5-ylmethyl)piperazin-1-yl]-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methanone

Example 40 was synthesized from example 20 following the deacetylationprocedure described in synthesis method B.

Yield: 12.6 mg (31.79 μmol, 89.7%), white solid.

LC/MS (ES-API): m/z=397.11 [M+H]⁺; calculated: 397.15; t_(R)1 (λ=220nm): 0.95 min; t_(R)2 (λ=220 nm): 0.99 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.82 (m, 2H, ArH), 6.75 (dd, J=1.4 Hz, 1H,ArH), 6.00 (s, 2H, O—CH₂—O), 6.49 (d, J=4.4 Hz, 1H, CH), 4.8-4.1 (m, 4H,4×OH), 3.6-3.2 (m, 4H, 4×CH), 3.0-2.8 (m, 8H, 4×NCH₂), 2.41 (m, 2H,NCH₂) ppm.

Example 41[(2S,3R,4S)-2,3-Diacetoxy-6-(4-butylpiperazine-1-carbonyl)-3,4-dihydro-2H-pyran-4-yl]acetate

Step 1: (2S,3R,4S)-2,3,4-triacetoxy-3,4-dihydro-2H-pyran-6-carboxylicacid

A solution of 2 g (5.52 mmol) 1,2,3,4-tetra-O-acetyl-β-D-glucuronic acidin 6 mL (63.59 mmol) acetic acid anhydride and 3 mL (22.08 mmol; 4 eq.)triethylamine was stirred at room temperature for 8 hours. The reactionmixture was diluted with water and lyophillized.

Yield: 1.6 g (5.38 mmol, 95.8%), colorless oil.

LC/MS (ES-API): not detectable.

¹H-NMR (400 MHz, CDCl₃): δ=7.99 (s, 1H, COOH), 6.33 (s, 1H, CH), 6.27(m, 1H, CH), 5.19 (m, 1H, CH), 5.10 (m, 1H, CH), 2.05 (s, 3H, CH₃), 2.04(s, 3H, CH₃), 2.03 (s, 3H, CH₃) ppm.

Method C Amide coupling with(2S,3R,4S)-2,3,4-triacetoxy-3,4-dihydro-2H-pyran-6-carboxylic acid(example 41, step 1)

To a solution of 0.66 mmol(2S,3R,4S)-2,3,4-triacetoxy-3,4-dihydro-2H-pyran-6-carboxylic acid(example 41, step 1) in 2 mL dimethylformamide were added 1.2 eq. (0.79mmol) HATU and 1.4 eq. (0.93 mmol) amine. The reaction mixture wasstirred at room temperature for 2 hours. The reaction was controlled byTLC and LC/MS. After completion the reaction mixture was extracted with5-10 mL dichloromethane. The organic phase was washed with 1 M aqueousHCl, water, saturated aqueous NaHCO₃ solution, and water. The organicphase was dried with Na₂SO₄, filtered, and evaporated. The crudematerial was purified by MPLC.

Step 2:[(2S,3R,4S)-2,3-diacetoxy-6-(4-butylpiperazine-1-carbonyl)-3,4-dihydro-2H-pyran-4-yl]acetate

Example 41 was synthesized from example 41, step 1 and1-n-butylpiperazine following the amide coupling procedure described insynthesis method C.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 2.2 0 4.8 2.8 4.8 4.8 6.0 4.810.010.1 6.0 10.1 10.1 9.1

Yield: 141 mg (0.331 mmol, 50.8%), orange oil.

LC/MS (ES-API): m/z=427.20 [M+H]⁺; calculated: 427.20; t_(R) (λ=220 nm):0.57 min (LC/MS—Method 1).

¹H-NMR (400 MHz, CDCl₃): δ=6.28 (d, J=3.6 Hz, 1H, C═CH), 5.67 (d, J=3.8Hz, 1H, CH), 5.25 (m, 1H, CH), 5.19 (m, 1H, CH), 3.73 (m, 4H, 2×NCH₂),2.60 (m, 4H, 2×NCH₂), 2.15 (s, 3H, CH₃), 2.13 (s, 3H, CH₃), 2.10 (s, 3H,CH₃) ppm.

Example 42[(2S,3R,4S)-2,3-Diacetoxy-6-(4-tert-butylpiperazine-1-carbonyl)-3,4-dihydro-2H-pyran-4-yl]acetate

Example 42 was synthesized from example 41, step 1 and1-tert-butylpiperazine following the amide coupling procedure describedin synthesis method C.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 2.2 0 4.8 2.8 4.8 4.8 6.0 4.810.010.1 6.0 10.1 10.1 9.1

Yield: 81 mg (0.190 mmol, 28.7%), orange oil.

LC/MS (ES-API): m/z=427.15 [M+H]⁺; calculated: 427.20; t_(R) (λ=220 nm):0.53 min (LC/MS—Method 1).

¹H-NMR (400 MHz, CDCl₃): δ=6.29 (d, J=3.6 Hz, 1H, C═CH), 5.69 (d, J=3.8Hz, 1H, CH), 5.24 (m, 1H, CH), 5.20 (m, 1H, CH), 3.75 (m, 4H, 2×NCH₂),2.62 (m, 4H, 2×NCH₂), 2.16 (s, 3H, CH₃), 2.13 (s, 3H, CH₃), 2.10 (s, 3H,CH₃), 1.27 (s, 9H, 3×CH₃) ppm.

Example 43[(2S,3R,4S)-2,3-Diacetoxy-6-[4-(cyclohexylmethyl)piperazine-1-carbonyl]-3,4-dihydro-2H-pyran-4-yl]acetate

Example 43 was synthesized from example 41, step 1 and1-cyclohexylmethylpiperazine following the amide coupling proceduredescribed in synthesis method C.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 2.2 0 4.8 2.8 4.8 4.8 6.0 4.810.010.1 6.0 10.1 10.1 9.1

Yield: 145 mg (0.311 mmol, 47.0%), orange oil.

LC/MS (ES-API): m/z=467.20 [M+H]⁺; calculated: 467.23; t_(R) (λ=220 nm):0.64 min (LC/MS—Method 1).

¹H-NMR (400 MHz, CDCl₃): δ=6.29 (d, J=3.2 Hz, 1H, C═CH), 5.63 (d, J=3.2Hz, 1H, CH), 5.24 (m, 1H, CH), 5.19 (m, 1H, CH), 3.75 (m, 4H, 2×NCH₂),2.62 (m, 4H, 2×NCH₂), 2.16 (s, 3H, CH₃), 2.13 (s, 3H, CH₃), 2.11 (s, 3H,CH₃), 1.73 (m, 1H, CH), 1.62 (m, 4H, 2×CH₂), 1.13 (m, 4H, 2×CH₂), 0.92(m, 2H, CH₂) ppm.

Example 44[(2S,3R,4S)-2,3-Diacetoxy-6-(4-phenylpiperazine-1-carbonyl)-3,4-dihydro-2H-pyran-4-yl]acetate

Example 44 was synthesized from example 41, step 1 and1-phenylpiperazine following the amide coupling procedure described insynthesis method C.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 2.2 0 4.8 2.8 4.8 4.8 6.0 4.810.010.1 6.0 10.1 10.1 9.1

Yield: 135 mg (0.303 mmol, 45.5%), orange oil.

LC/MS (ES-API): m/z=447.13 [M+H]⁺; calculated: 447.17; t_(R) (λ=220 nm):0.81 min (LC/MS—Method 1).

¹H-NMR (400 MHz, CDCl₃): δ=7.31 (t, J=7.9 Hz, 2H, ArH), 7.00 (m, 3H,ArH), 6.32 (dd, J=0.9 Hz, 1H, C═CH), 5.67 (dd, J=0.8 Hz, 1H, CH), 5.27(m, 1H, CH), 5.21 (m, 1H, CH), 3.76 (m, 4H, 2×NCH₂), 3.22 (m, 4H,2×NCH₂), 2.15 (s, 3H, CH₃), 2.13 (s, 3H, CH₃), 2.10 (s, 3H, CH₃) ppm.

Example 45[(2S,3R,4S)-2,3-Diacetoxy-6-[(4-phenylpiperazin-1-yl)methyl]-3,4-dihydro-2H-pyran-4-yl]acetate

Step 1: [(2S,3R,4S)-2,3-diacetoxy-6-formyl-3,4-dihydro-2H-pyran-4-yl]acetate

A solution of 2.87 mL dry DMSO (40.31 mmol; 2.6 eq.) in 3 mL drydichloromethane was dropped slowly to a solution of 1.61 mLoxalylchloride (18.60 mmol; 1.2 eq.) in 4 mL dry dichloromethane at −70°C. After stirring the reaction mixture at −70° C. for 30 minutes, asolution of 5.4 g 1,2,3,4-tetra-O-acetyl-β-D-glucopyranose in 20 mL drydichloromethane was added. After stirring the reaction mixture at −70°C. for 30 minutes, 11 mL triethylamine were added slowly. The reactionmixture was warmed to room temperature and diluted with 20 mL water.After stirring for 10 minutes at room temperature, the aqueous phase wasseparated and extracted with dichloromethane. The combined organicphases were dried with Na₂SO₄, filtered, and evaporated.

Yield: 3.13 g (10.94 mmol, 70.5%), colorless oil.

TLC: R_(f)=0.509 (ethylacetate/n-heptane, 2:1).

LC/MS (ES-API): m/z=304.05 [M+H₂O]+; calculated: 287.07; t_(R) (λ=254nm): 0.63 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=9.28 (s, 1H, CHO), 5.19 (d, J=4.2 Hz, 1H,CH), 5.10 (m, 2H, 2×CH), 2.08 (s, 3H, CH₃), 2.07 (s, 3H, CH₃), 2.04 (s,3H, CH₃) ppm.

Method D Reductive amination with[(2S,3R,4S)-2,3-diacetoxy-6-formyl-3,4-dihydro-2H-pyran-4-yl] acetate(example 45, step 1)

To a solution of 0.52 mmol[(2S,3R,4S)-2,3-diacetoxy-6-formyl-3,4-dihydro-2H-pyran-4-yl] acetate(example 45, step 1) in 10 mL dichloroethane were added 0.57 mmol (1.1eq.) amine and 0.74 mmol (1.41 eq.) sodiumtriacetoxyboronhydride. Thereaction mixture was stirred at room temperature over night. Thereaction was controlled by TLC and LC/MS. The reaction mixture wasfiltered and evaporated. The crude material was purified by MPLC.

Step 2:[(2S,3R,4S)-2,3-diacetoxy-6-[(4-phenylpiperazin-1-yl)methyl]-3,4-dihydro-2H-pyran-4-yl]acetate

Example 45 was synthesized from example 45, step 1 and1-phenylpiperazine following the reductive amidation procedure describedin synthesis method D.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 254 nm; eluent:(A) n-heptane, (B) ethylacetate.

Yield: 154 mg (0.356 mmol, 68.0%), yellow oil.

TLC: R_(f)=0.639 (ethylacetate/n-heptane, 2:1).

LC/MS (ES-API): m/z=433.20 [M+H]⁺; calculated: 433.19; t_(R) (λ=220 nm):1.26 min (LC/MS—Method 1).

MPLC Gradient

start % B end % B duration [min] 0.0 0.0 1.6 0.0 70.2 28.0

¹H-NMR (400 MHz, DMSO-d₆): δ=7.20 (t, J=8.0 Hz, 2H, ArH), 6.92 (d, J=8.0Hz, 2H, ArH), 6.76 (d, J=7.2 Hz, 1H, ArH), 5.96 (dd, J=3.6 Hz, J=1.0 Hz1H, CH), 5.12 (d, J=4.2 Hz, 1H, CH), 5.04 (m, 2H, 2×CH), 3.12 (m, 4H,2×NCH₂), 3.02 (AB-system, q, J=14.3 Hz, 2H, CH₂), 2.50 (m, 4H, 2×NCH₂),2.08 (s, 3H, CH₃), 2.07 (s, 3H, CH₃), 2.04 (s, 3H, CH₃) ppm.

¹³C-NMR (150 MHz, DMSO-d₆): δ=169.83 (s, C), 168.62 (s, C), 168.40 (s,C), 151.31 (s, 2×C), 129.16 (s, CH), 118.98 (s, CH), 115.23 (s, CH),97.54 (s, CH), 93.66 (s, C), 88.92 (s, CH), 66.82 (s, CH), 64.19 (s,CH), 52.04 (s, 2×CH), 47.96 (s, 2×CH), 21.13 (s, CH₃), 20.85 (s, CH₃),20.74 (s, CH₃) ppm.

Example 46[(2S,3R,4S)-2,3-Diacetoxy-6-[[4-[(E)-cinnamyl]piperazin-1-yl]methyl]-3,4-dihydro-2H-pyran-4-yl]acetate

Example 46 was synthesized from example 45, step 1 andtrans-1-cinnamylpiperazine following the reductive amidation proceduredescribed in synthesis method D.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

Yield: 194 mg (0.411 mmol, 78.3%), yellow oil.

TLC: R_(f)=0.136 (ethylacetate/n-heptane, 2:1).

LC/MS (ES-API): m/z=473.23 [M+H]⁺; calculated: 473.22; t_(R) (λ=220 nm):1.32 min (LC/MS—Method 1).

MPLC Gradient

start % B end % B duration [min] 0.0 0.0 1.3 0.0 13.3 15.1

¹H-NMR (400 MHz, DMSO-d₆): δ=7.42 (d, J=7.3 Hz, 2H, ArH), 7.31 (t, J=7.3Hz, 2H, ArH), 7.23 (t, J=7.3 Hz, 1H, ArH), 6.52 (d, J=15.9 Hz, 1H, CH),6.27 (m, 1H, CH), 6.18 (dd, J=1.2 Hz, 1H, CH), 5.08 (d, J=4.1 Hz, 1H,CH), 5.03 (m, 2H, 2×CH), 3.33 (m, 6H, 3×NCH₂), 2.96 (q, J=14.2 Hz, 2H,CH₂), 2.41 (m, 4H, 2×NCH₂), 2.08 (s, 3H, CH₃), 2.07 (s, 3H, CH₃), 2.04(s, 3H, CH₃) ppm.

Example 47[(2S,3R,4S)-2,3-Diacetoxy-6-[[4-(1,3-benzodioxol-5-ylmethyl)piperazin-1-yl]methyl]-3,4-dihydro-2H-pyran-4-yl]acetate

Example 47 was synthesized from example 45, step 1 and1-(1,3-benzodioxol-5-ylmethyl)piperazine following the reductiveamidation procedure described in synthesis method D.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

Yield: 249 mg (0.508 mmol, 96.9%), yellow oil.

TLC: R_(f)=0.242 (ethylacetate/n-heptane, 2:1).

LC/MS (ES-API): m/z=491.24 [M+H]⁺; calculated: 491.19; t_(R) (λ=220 nm):1.19 min (LC/MS—Method 1).

MPLC Gradient

start % B end % B duration [min] 0.0 0.0 1.9 0.0 10.1 13.1

¹H-NMR (400 MHz, DMSO-d₆): δ=6.84 (m, 2H, ArH), 6.73 (dd, J=1.4 Hz, 1H,ArH), 6.28 (dd, J=1.0 Hz, 1H, CH), 5.97 (s, 2H, O—CH₂—O), 5.08 (d, J=4.2Hz, 1H, CH), 5.04 (m, 2H, 2×CH), 3.31 (m, 4H, 2×NCH₂), 2.94 (q, J=14.4Hz, 2H, CH₂), 2.38 (m, 4H, 2×NCH₂), 2.08 (s, 3H, CH₃), 2.07 (s, 3H,CH₃), 2.03 (s, 3H, CH₃) ppm.

Example 48[(2S,3R,4S)-2,3-Diacetoxy-6-[[4-(4-chlorophenyl)piperazin-1-yl]methyl]-3,4-dihydro-2H-pyran-4-yl]acetate

Example 48 was synthesized from example 45, step 1 and1-(4-chlorophenyl)piperazine following the reductive amidation proceduredescribed in synthesis method D.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 220 nm; eluent:(A) dichloromethane, (B) ethanol.

Yield: 131 mg (0.281 mmol, 53.5%), yellow oil.

TLC: R_(f)=0.636 (ethylacetate/n-heptane, 2:1).

LC/MS (ES-API): m/z=467.16 [M+H]⁺; calculated: 467.91; t_(R) (λ=220 nm):1.40 min (LC/MS—Method 1).

MPLC Gradient

start % B end % B duration [min] 0.0 0.0 2.0 0.0 10.1 23.0

¹H-NMR (400 MHz, DMSO-d₆): δ=7.21 (d, J=9.1 Hz, 2H, ArH), 6.93 (d, J=9.1Hz, 2H, ArH), 6.20 (dd, J=1.1 Hz, 1H, CH), 5.12 (d, J=4.4 Hz, 1H, CH),5.04 (m, 2H, 2×CH), 3.12 (m, 4H, 2×NCH₂), 3.02 (q, J=14.1 Hz, 2H, CH₂),2.51 (m, 4H, 2×NCH₂), 2.07 (s, 3H, CH₃), 2.06 (s, 3H, CH₃), 2.04 (s, 3H,CH₃) ppm.

Example 49[(2S,3R,4S)-2,3-Diacetoxy-6-[[4-(2-chlorophenyl)piperazin-1-yl]methyl]-3,4-dihydro-2H-pyran-4-yl]acetate

Example 49 was synthesized from example 45, step 1 and1-(2-chlorophenyl)piperazine following the reductive amidation proceduredescribed in synthesis method D.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 254 nm; eluent:(A) n-heptane, (B) ethylacetate.

Yield: 144 mg (0.308 mmol, 58.9%), yellow oil.

TLC: R_(f)=0.621 (ethylacetate/n-heptane, 2:1).

LC/MS (ES-API): m/z=467.16 [M+H]⁺; calculated: 467.91; t_(R) (λ=220 nm):1.38 min (LC/MS—Method 1).

MPLC Gradient

start % B end % B duration [min] 0.0 0.0 1.6 0.0 70.2 23.0

¹H-NMR (400 MHz, DMSO-d₆): δ=7.39 (d, J=7.9 Hz, 1H, ArH), 7.28 (t, J=7.7Hz, 1H, ArH), 7.17 (d, J=8.1 Hz, 1H, ArH), 7.03 (t, J=7.6 Hz, 1H, ArH),6.21 (dd, J=1.2 Hz, 1H, CH), 5.13 (d, J=4.1 Hz, 1H, CH), 5.04 (m, 2H,2×CH), 3.07 (q, J=14.4 Hz, 2H, CH₂), 2.98 (m, 4H, 2×NCH₂), 2.59 (m, 4H,2×NCH₂), 2.08 (s, 3H, CH₃), 2.07 (s, 3H, CH₃), 2.03 (s, 3H, CH₃) ppm.

Example 50[(2S,3R,4S)-2,3-Diacetoxy-6-[[4-(4-methoxyphenyl)piperazin-1-yl]methyl]-3,4-dihydro-2H-pyran-4-yl]acetate

Example 50 was synthesized from example 45, step 1 and1-(4-methoxyphenyl)piperazine following the reductive amidationprocedure described in synthesis method D.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 254 nm; eluent:(A) n-heptane, (B) ethylacetate.

Yield: 151 mg (0.326 mmol, 62.3%), yellow oil.

TLC: R_(f)=0.530 (ethylacetate/n-heptane, 2:1).

LC/MS (ES-API): m/z=463.19 [M+H]⁺; calculated: 463.20; t_(R) (λ=220 nm):1.25 min (LC/MS—Method 1).

MPLC Gradient

start % B end % B duration [min] 0.0 0.0 1.3 0.0 100.0 13.7 100.0 100.03.6

¹H-NMR (400 MHz, DMSO-d₆): δ=6.88 (d, J=9.1 Hz, 2H, ArH), 6.80 (d, J=9.1Hz, 2H, ArH), 6.20 (dd, J=1.0 Hz, 1H, CH), 5.12 (d, J=4.1 Hz, 1H, CH),5.04 (m, 2H, 2×CH), 3.69 (s, 3H, OCH₃), 3.00 (m, 6H, CH₂+NCH₂), 2.52 (m,4H, NCH₂), 2.08 (s, 3H, CH₃), 2.07 (s, 3H, CH₃), 2.04 (s, 3H, CH₃) ppm.

Example 51[(2S,3R,4S)-2,3-Diacetoxy-6-[[4-(3-methoxyphenyl)piperazin-1-yl]methyl]-3,4-dihydro-2H-pyran-4-yl]acetate

Example 51 was synthesized from example 45, step 1 and1-(3-methoxyphenyl)piperazine following the reductive amidationprocedure described in synthesis method D.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 254 nm; eluent:(A) n-heptane, (B) ethylacetate.

Yield: 135 mg (0.292 mmol, 55.7%), yellow oil.

TLC: R_(f)=0.591 (ethylacetate/n-heptane, 2:1).

LC/MS (ES-API): m/z=463.24 [M+H]⁺; calculated: 463.20; t_(R) (λ=220 nm):1.28 min (LC/MS—Method 1).

MPLC Gradient

start % B end % B duration [min] 0.0 0.0 1.3 0.0 100.0 13.7 100.0 100.03.6

¹H-NMR (400 MHz, DMSO-d₆): δ=7.09 (t, J=8.2 Hz, 1H, ArH), 6.51 (d, J=8.2Hz, 1H, ArH), 6.43 (s, 1H, ArH), 6.35 (dd, J=1.1 Hz, 1H, CH), 5.12 (d,J=4.3 Hz, 1H, CH), 5.04 (m, 1H, CH), 3.71 (s, 3H, OCH₃), 3.11 (m, 4H,2×NCH₂), 3.01 (q, J=14.3 Hz, 2H, CH₂), 2.52 (m, 4H, 2×NCH₂), 2.08 (s,3H, CH₃), 2.07 (s, 3H, CH₃), 2.04 (s, 3H, CH₃) ppm.

Example 52[(2S,3R,4S)-2,3-Diacetoxy-6-[[4-(2-methoxyphenyl)piperazin-1-yl]methyl]-3,4-dihydro-2H-pyran-4-yl]acetate

Example 52 was synthesized from example 45, step 1 and1-(2-methoxyphenyl)piperazine following the reductive amidationprocedure described in synthesis method D.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 254 nm; eluent:(A) n-heptane, (B) ethylacetate.

Yield: 167 mg (0.361 mmol, 68.9%), yellow oil.

TLC: R_(f)=0.533 (ethylacetate/n-heptane, 2:1).

LC/MS (ES-API): m/z=463.24 [M+H]⁺; calculated: 463.20; t_(R) (λ=220 nm):1.26 min (LC/MS—Method 1).

MPLC Gradient

start % B end % B duration [min] 0.0 0.0 1.3 0.0 100.0 13.7 100.0 100.03.6

¹H-NMR (400 MHz, DMSO-d₆): δ=6.90 (m, 4H, ArH), 6.21 (dd, J=1.0 Hz, 1H,CH), 5.12 (d, J=4.4 Hz, 1H, CH), 5.03 (m, 1H, CH), 3.68 (s, 3H, OCH₃),3.04 (q, J=14.3 Hz, 2H, CH₂), 2.96 (m, 4H, 2×NCH₂), 2.54 (m, 4H,2×NCH₂), 2.10 (s, 3H, CH₃), 2.09 (s, 3H, CH₃), 2.05 (s, 3H, CH₃) ppm.

Example 53[(2S,3R,4S)-2,3-Diacetoxy-6-[[4-(4-bromophenyl)piperazin-1-yl]methyl]-3,4-dihydro-2H-pyran-4-yl]acetate

Example 53 was synthesized from example 45, step 1 and1-(4-bromophenyl)piperazine following the reductive amidation proceduredescribed in synthesis method D.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica12 g; flow rate: 30 mL/min; wavelength for detection: 254 nm; eluent:(A) n-heptane, (B) ethylacetate.

Yield: 248 mg (0.485 mmol, 92.5%), yellow oil.

TLC: R_(f)=0.644 (ethylacetate/n-heptane, 2:1).

LC/MS (ES-API): m/z=511.14 [M+H]⁺; calculated: 511.10; t_(R) (λ=220 nm):1.42 min (LC/MS—Method 1).

MPLC Gradient

start % B end % B duration [min] 0.0 0.0 1.3 0.0 100.0 13.7 100.0 100.03.6

¹H-NMR (400 MHz, DMSO-d₆): δ=7.33 (d, J=9.0 Hz, 2H, ArH), 6.88 (d, J=9.0Hz, 2H, ArH), 6.20 (dd, J=1.2 Hz, 1H, CH), 5.12 (d, J=4.3 Hz, 1H, CH),5.04 (m, 2H, 2×CH), 3.12 (m, 4H, 2×NCH₂), 3.03 (q, J=14.2 Hz, 2H, CH₂),2.52 (m, 4H, 2×NCH₂), 2.09 (s, 3H, CH₃), 2.08 (s, 3H, CH₃), 2.05 (s, 3H,CH₃) ppm.

Example 545-(Dimethylamino)-N-[[1-[(3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]triazol-4-yl]methyl]naphthalene-1-sulfonamide

Step 1: 5-(dimethylamino)-N-prop-2-ynyl-naphthalene-1-sulfonamide

To a solution of 405 mg (1.5 mmol) dansylchloride in 4 mLdimethylformamide were added 91 mg (1.65 mmol) propargylamine and 530 μl(3 mmol, 2 eq.) N,N-diisopropylethylamine. The reaction mixture wasstirred at 100° C. in the microwave for 45 minutes. The reaction mixturewas filtered and evaporated.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica24 g Gold; flow rate: 40 mL/min; wavelength for detection: 254 nm;eluent: (A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0.0 0.0 2.0 0.0 50.0 33.0

Yield: 92 mg (0.319 mmol, 21.5%), yellow oil.

TLC: R_(f)=0.510 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=289.13 [M+H]⁺; calculated: 289.09; t_(R) (λ=220 nm):1.62 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.48 (d, J=8.6 Hz, 1H, ArH), 8.40 (s, 1H,SO₂NH), 8.25 (d, J=8.6 Hz, 1H, ArH), 8.13 (dd, J=1.1 Hz, 1H, ArH), 7.60(m, 2H, ArH), 7.26 (d, J=7.4 Hz, 1H, ArH), 3.71 (s, 2H, NCH₂), 2.91 (s,1H, CH), 2.84 (s, 6H, 2×NCH₃) ppm.

Method E

Copper-Catalyzed Azide-Alkyne-Cycloaddition (CuAAC) withAzidodeoxyglucose

To a solution of 146 μmol azidodeoxyglucose (1-azido-1-deoxyglucose,2-azido-2-deoxyglucose, or 6-azido-6-deoxyglucose) in 0.5 mL water 1.1eq. alkyne were added. If needed dimethylformamide was added until thereaction mixture was a clear solution. A mixture of 0.1 eq. CuSO₄*5 H₂O(0.1 M in water), 0.25 eq. sodium ascorbate (1 M in water), and 0.4 eq.THPTA (0.5 M in water) was added. The reaction mixture was stirred atroom temperature for 2-6 hours. The reaction was controlled by TLC andLC/MS. The reaction mixture was evaporated and purified by HPLC.Alpha/beta isomers were not separated, NMR data belong to only oneisomer.

Purification: Agilent 1200 preparative HPLC; column: Agilent Prep-C₁₈column (10 μm, 21.5×150 mm); flow rate: 40 mL/min; wavelength fordetection: 220 nm; 254 nm; 324 nm; eluent: (A) water, (B) acetonitrile.

HPLC-Gradient

start % B end % B duration [min] 3.0 3.0 5.0 3.0 90.0 7.5 90.0 90.0 2.590.0 10.0 0.5 10.0 10.0 2.0

Step 2:5-(dimethylamino)-N-[[1-[(3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)-tetrahydropyran-3-yl]triazol-4-yl]methyl]naphthalene-1-sulfonamide

Example 54 was synthesized from 2-azido-2-deoxyglucose and example 45,step 1 following the CuAAC procedure described in synthesis method E.

Yield: 33 mg (67 μmol, 46.7%), yellow oil.

TLC: R_(f)=0.032 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=494.19 [M+H]⁺; calculated: 494.16; t_(R)1 (λ=220nm): 1.17 min; t_(R)2 (λ=220 nm): 1.20 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.47 (d, J=8.6 Hz, 2H, ArH), 8.40 (s, 1H,SNH), 8.13 (d, J=7.3 Hz, 1H, ArH), 7.84 (s, 1H, NCH), 7.61 (m, 2H, ArH),7.27 (d, J=7.4 Hz, 1H, ArH), 6.95 (d, J=6.4 Hz, 1H, OH), 5.17 (d, J=6.2Hz, 1H, OH), 5.09 (d, J=6.2 Hz, 1H, OH), 4.89 (dd, J=6.4 Hz, 1H, CH),4.61 (dd, J=6.4 Hz, 1H, OH), 4.07 (s, 2H, NCH₂), 4.01 (m, 1H, CH), 3.85(m, 1H, CH), 3.74 (m, 1H, CH), 3.53 (m, 2H, CH₂), 3.22 (m, 1H, CH₂),2.84 (s, 6H, 2×NCH₃) ppm.

Example 55(3R,4R,5S,6R)-6-(Hydroxymethyl)-3-(4-phenyltriazol-1-yl)tetrahydropyran-2,4,5-triol

Example 55 was synthesized from 2-azido-2-deoxyglucose andethynylbenzene following the CuAAC procedure described in synthesismethod E.

Yield: 15 mg (49 μmol, 33.4%), white solid.

TLC: R_(f)=0.333 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=308.10 [M+H]⁺; calculated: 308.12; t_(R)1 (λ=220nm): 0.81 min; t_(R)2 (λ=220 nm): 0.85 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.61 (s, 1H, NCH), 7.86 (dd, J=1.3 Hz, 2H,ArH), 7.45 (m, 2H, ArH), 7.40 (m, 1H, ArH), 7.10 (d, J=6.2 Hz, 1H, OH),5.48 (d, J=6.2 Hz, 1H, OH), 5.25 (d, J=6.6 Hz, 1H, OH), 5.25 (t, J=4.2Hz, 1H, CH), 4.65 (t, J=6.1 Hz, 1H, OH), 4.65 (dd, J=10.8 Hz, J=3.1 Hz,1H, CH), 4.08 (m, 1H, CH), 3.96 (m, 1H, OH), 3.76 (m, 1H, CH₂), 3.52 (m,1H, CH₂), 3.36 (m, 1H, CH), 3.29 (m, 1H, CH) ppm.

¹³C-NMR (150 MHz, DMSO-d₆): δ=145.70 (s, C), 131.00 (s, C), 128.90 (s,CH), 127.64 (s, CH), 125.05 (s, CH), 120.64 (s, CH), 90.80 (s, CH),72.42 (s, CH), 70.93 (s, CH), 69.72 (s, CH), 65.24 (s, CH), 60.86 (s,CH₂) ppm.

Example 56(3R,4R,5S,6R)-6-(Hydroxymethyl)-3-[4-(p-tolyl)triazol-1-yl]tetrahydropyran-2,4,5-triol

Example 56 was synthesized from 2-azido-2-deoxyglucose and1-ethynyl-4-methylbenzene following the CuAAC procedure described insynthesis method E.

Yield: 34 mg (106 μmol, 72.4%), white solid.

TLC: R_(f)=0.349 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=322.19 [M+H]⁺; calculated: 322.13; t_(R)1 (λ=220nm): 1.07 min; t_(R)2 (λ=220 nm): 1.11 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.47 (s, 1H, NCH), 7.76 (d, J=8.1 Hz, 2H,ArH), 7.24 (d, J=8.1 Hz, 2H, ArH), 6.93 (d, J=4.4 Hz, 1H, OH), 5.29 (d,J=5.4 Hz, 1H, OH), 5.20 (d, J=5.4 Hz, 1H, OH), 5.00 (dd, J=8.0 Hz, 1H,CH), 4.56 (dd, J=3.2 Hz, 1H, OH), δ=4.09 (m, 1H, CH), 3.96 (m, 1H, CH),3.76 (m, 1H, CH₂), 3.53 (m, 1H, CH₂), 3.36 (m, 1H, CH), 3.28 (m, 1H,CH), 2.35 (s, 3H, CH₃) ppm.

Example 57(3R,4R,5S,6R)-6-(Hydroxymethyl)-3-[4-(3-phenylpropyl)triazol-1-yl]tetrahydropyran-2,4,5-triol

Example 57 was synthesized from 2-azido-2-deoxyglucose andpent-4-yn-1-ylbenzene following the CuAAC procedure described insynthesis method E.

Yield: 40 mg (114 μmol, 78.3%), white solid.

TLC: R_(f)=0.406 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=350.19 [M+H]⁺; calculated: 350.16; t_(R)1 (λ=220nm): 1.24 min; t_(R)2 (λ=220 nm): 1.26 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.78 (s, 1H, NCH), 7.75 (m, 5H, ArH), 6.81(d, J=5.0 Hz, 1H, OH), 5.22 (d, J=5.7 Hz, 1H, OH), 5.12 (d, J=5.7 Hz,1H, OH), 4.92 (dd, J=6.7 Hz, 1H, CH), 4.52 (dd, J=5.5 Hz, 1H, OH), 4.09(m, 1H, CH), 3.95 (m, 1H, CH), 3.76 (m, 1H, CH₂), 3.53 (m, 1H, CH₂),3.36 (m, 1H, CH), 3.28 (m, 1H, CH), 2.61 (m, 4H, CH₂), 1.90 (m, 2H, CH₂)ppm.

Example 58 Methyl4-[1-[(3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]triazol-4-yl]benzoate

Example 58 was synthesized from 2-azido-2-deoxyglucose and4-ethynylmethylbenzoate following the CuAAC procedure described insynthesis method E.

Yield: 31 mg (85 μmol, 58.0%), white solid.

TLC: R_(f)=0.429 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=366.12 [M+H]⁺; calculated: 366.12; t_(R)1 (λ=220nm): 0.97 min; t_(R)2 (λ=220 nm): 1.00 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.80 (s, 1H, NCH), 8.05 (d, J=8.2 Hz, 2H,ArH), 8.00 (d, J=8.2 Hz, 2H, ArH), 7.09 (d, J=6.4 Hz, 1H, OH), 5.49 (d,J=6.3 Hz, 1H, OH), 5.25 (d, J=5.7 Hz, 1H, OH), 5.00 (dd, J=8.0 Hz, 1H,CH), 4.65 (dd, J=5.2 Hz, 1H, OH), 4.09 (m, 1H, CH), 3.95 (m, 1H, CH),3.87 (s, 3H, OCH₃), 3.76 (m, 1H, CH₂), 3.53 (m, 1H, CH₂), 3.36 (m, 1H,CH), 3.28 (m, 1H, CH) ppm.

Example 59(3R,4R,5S,6R)-3-[4-[[Benzyl(methyl)amino]methyl]triazol-1-yl]-6-(hydroxymethyl)tetrahydropyran-2,4,5-triol

Example 59 was synthesized from 2-azido-2-deoxyglucose andN-benzyl-N-methylprop-2-yn-1-amine following the CuAAC proceduredescribed in synthesis method E.

Yield: 32 mg (88 μmol, 60.1%), white solid.

TLC: R_(f)=0.064 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=365.20 [M+H]⁺; calculated: 365.17; t_(R) (ELSD):0.34 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.00 (s, 1H, NCH), 7.71 (m, 5H, ArH), 6.82(d, J=6.0 Hz, 1H, OH), 5.25 (d, J=5.8 Hz, 1H, OH), 5.14 (d, J=5.8 Hz,1H, OH), 4.93 (dd, J=5.7 Hz, 1H, CH), 4.49 (dd, J=5.2 Hz, 1H, OH), 4.08(m, 1H, CH), 3.95 (m, 1H, CH), 3.76 (m, 2H, CH₂), 3.62 (s, 3H, NCH₃),3.36 (m, 1H, CH), 3.28 (m, 1H, CH), 2.13 (m, 4H, 2×NCH₂) ppm.

Example 60(3R,4R,5S,6R)-6-(Hydroxymethyl)-3-[4-(6-methoxy-2-naphthyl)triazol-1-yl]tetrahydropyran-2,4,5-triol

Example 60 was synthesized from 2-azido-2-deoxyglucose and2-ethynyl-6-methoxynaphthalene following the CuAAC procedure describedin synthesis method E.

Yield: 28 mg (72 μmol, 49.4%), white solid.

TLC: R_(f)=0.461 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=388.16 [M+H]⁺; calculated: 388.14; t_(R)1 (λ=220nm): 1.27 min; t_(R)2 (λ=220 nm): 1.29 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.64 (s, 1H, NCH), 7.88 (m, 4H, ArH), 7.34(s, 1H, ArH), 7.07 (d, J=6.5 Hz, 1H, OH), 5.47 (d, J=6.4 Hz, 1H, OH),5.23 (d, J=6.4 Hz, 1H, OH), 5.00 (dd, J=6.6 Hz, 1H, CH), 4.49 (dd, J=5.2Hz, 1H, OH), 4.11 (m, 1H, CH), 3.98 (m, 1H, CH), 3.89 (s, 3H, OCH₃),3.77 (m, 1H, CH₂), 3.53 (m, 1H, CH₂), 3.46 (m, 1H, CH), 3.38 (m, 1H, CH)ppm.

Example 61(3R,4R,5S,6R)-3-[4-[4-Chloro-6-methyl-2-(p-tolyl)pyrimidin-5-yl]triazol-1-yl]-6-(hydroxymethyl)tetrahydropyran-2,4,5-triol

Example 61 was synthesized from 2-azido-2-deoxyglucose and4-chloro-5-ethynyl-6-methyl-2-(p-tolyl)pyrimidine following the CuAACprocedure described in synthesis method E.

Yield: 51 mg (110 μmol, 75.5%), white solid.

TLC: R_(f)=0.556 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=462.22 [M+H]⁺; calculated: 462.15; t_(R)1 (λ=220nm): 1.51 min; t_(R)2 (λ=220 nm): 1.52 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.19 (d, J=8.2 Hz, 2H, ArH), 7.89 (s, 1H,NCH), 7.71 (d, J=8.2 Hz, 2H, ArH), 6.93 (d, J=6.6 Hz, 1H, OH), 5.21 (d,J=6.1 Hz, 1H, OH), 5.14 (d, J=6.1 Hz, 1H, OH), 4.91 (dd, J=6.7 Hz, 1H,CH), 4.50 (dd, J=5.5 Hz, 1H, OH), 4.21 (s, 3H, CH₃), 4.05 (m, 1H, CH),3.97 (m, 1H, CH), 3.85 (m, 2H, CH₂), 3.71 (m, 1H, CH), 3.50 (m, 1H, CH),2.62 (s, 3H, CH₃) ppm.

Example 62Ethyl-2-diethoxyphosphoryl-3-[1-[(3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)-tetrahydropyran-3-yl]triazol-4-yl]propanoate

Example 62 was synthesized from 2-azido-2-deoxyglucose and ethyl2-diethoxyphosphorylpent-4-ynoate following the CuAAC proceduredescribed in synthesis method E.

Yield: 30 mg (64 μmol, 43.9%), white solid.

TLC: R_(f)=0.389 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=468.17 [M+H]⁺; calculated: 468.17; t_(R)1 (λ=220nm): 0.99 min; t_(R)2 (λ=220 nm): 1.01 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.89 (s, 1H, NCH), 6.90 (d, J=6.6 Hz, 1H,OH), 5.17 (d, J=6.0 Hz, 1H, OH), 5.10 (d, J=6.0 Hz, 1H, OH), 4.86 (dd,J=6.5 Hz, 1H, CH), 4.61 (dd, J=5.5 Hz, 1H, OH), 4.01 (m, 1H, CH), 3.88(m, 1H, CH), 3.61 (m, 1H, CH), 3.50 (m, 1H, CH₂), 3.22 (m, 1H, CH₂),3.01 (m, 1H, CH), 1.29 (m, 9H, CH₃), 1.14 (m, 6H, CH₂) ppm.

Example 63(3R,4R,5S,6R)-6-(Hydroxymethyl)-3-[4-[[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]methyl]triazol-1-yl]tetrahydropyran-2,4,5-triol

Step 1: 4-nitro-N-prop-2-ynyl-2,1,3-benzoxadiazol-7-amine

To a solution of 450 mg (2.23 mmol)4-chloro-7-nitro-2,1,3-benzoxadiazole in 4 mL dry dimethylformamide wereadded 135 mg (2.46 mmol) propargylamine and 395 μl (2.23 mmol)N,N-diisopropylethylamine. The reaction mixture was stirred at 100° C.in the microwave for 45 minutes and evaporated.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica40 g; flow rate: 40 mL/min; wavelength for detection: 254 nm; eluent:(A) n-heptane, (B) ethylacetate.

MPLC Gradient

start % B end % B duration [min] 0.0 0.0 1.6 0.0 27.7 16.6 27.7 79.819.9 79.8 0.0 0.0 0.0 0.0 2.9

Yield: 98 mg (0.449 mmol, 20.1%), orange solid.

TLC: R_(f)=0.664 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=219.01 [M+H]⁺; calculated: 219.04; t_(R) (λ=220 nm):1.32 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=9.62 (s, 1H, NH), 8.62 (d, J=8.7 Hz, 1H,ArH), 6.48 (d, J=8.7 Hz, 1H, ArH), 4.34 (s, 2H, NCH₂), 3.37 (s, 1H, CH)ppm.

Step 2:(3R,4R,5S,6R)-6-(hydroxymethyl)-3-[4-[[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]methyl]triazol-1-yl]tetrahydropyran-2,4,5-triol

Example 63 was synthesized from 2-azido-2-deoxyglucose and example 63,step 1 following the CuAAC procedure described in synthesis method E.

Yield: 43 mg (101 μmol, 69.5%), orange solid.

TLC: R_(f)=0.294 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=424.10 [M+H]⁺; calculated: 424.16; t_(R)1 (λ=220nm): 0.77 min; t_(R)2 (λ=220 nm): 0.81 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=9.83 (s, 1H, NH) 8.62 (d, J=8.7 Hz, 1H,ArH), 8.04 (s, 1H, NCH), 6.93 (d, J=6.4 Hz, 1H, OH), 6.51 (d, J=8.7 Hz,1H, ArH), 5.21 (d, J=6.2 Hz, 1H, OH), 5.14 (d, J=6.2 Hz, 1H, OH), 4.92(dd, J=6.4 Hz, 1H, CH), 4.67 (s, 2H, NCH₂), 4.61 (dd, J=6.4 Hz, 1H, OH),4.06 (m, 1H, CH), 3.90 (m, 1H, CH), 3.64 (m, 1H, CH), 3.52 (m, 2H, CH₂),3.22 (m, 1H, CH₂), 3.01 (m, 1H, CH) ppm.

Example 64(3R,4R,5S,6R)-3-[4-(Cyclohexylmethyl)triazol-1-yl]-6-(hydroxymethyl)tetrahydropyran-2,4,5-triol

Example 64 was synthesized from 2-azido-2-deoxyglucose andprop-2-yn-1-ylcyclohexane following the CuAAC procedure described insynthesis method E.

Yield: 23 mg (70 μmol, 48.0%), white solid.

TLC: R_(f)=0.437 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=328.24 [M+H]⁺; calculated: 328.18; t_(R)1 (λ=220nm): 1.23 min; t_(R)2 (λ=220 nm): 1.26 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.67 (s, 1H, NCH), 6.78 (d, J=6.5 Hz, 1H,OH), 5.19 (d, J=6.3 Hz, 1H, OH), 5.14 (d, J=5.7 Hz, 1H, OH), 4.90 (dd,J=8.0 Hz, 1H, CH), 4.52 (dd, J=6.2 Hz, 1H, OH), 4.41 (dd, J=4.2 Hz, 1H,OH), 4.07 (m, 1H, CH), 3.76 (m, 2H, CH₂), 3.56 (m, 2H, 2×CH), 3.20 (m,1H, CH), 1.60 (m, 6H, CH₂), 1.55 (m, 1H, CH), 1.21 (m, 4H, CH₂), 0.93(m, 2H, CH₂) ppm.

Example 65(3R,4R,5S,6R)-6-(Hydroxymethyl)-3-(4-pentyltriazol-1-yl)tetrahydropyran-2,4,5-triol

Example 65 was synthesized from 2-azido-2-deoxyglucose and hept-1-ynefollowing the CuAAC procedure described in synthesis method E.

Yield: 19 mg (63 μmol, 43.1%), white solid.

TLC: R_(f)=0.405 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=302.22 [M+H]⁺; calculated: 302.16; t_(R)1 (λ=220nm): 1.08 min; t_(R)2 (λ=220 nm): 1.12 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.73 (s, 1H, NCH), 6.81 (d, J=4.5 Hz, 1H,OH), 5.21 (d, J=5.7 Hz, 1H, OH), 5.16 (d, J=5.6 Hz, 1H, OH), 4.91 (dd,J=6.7 Hz, 1H, CH), 4.52 (dd, J=5.9 Hz, 1H, OH), 4.39 (dd, J=3.2 Hz, 1H,OH), 4.04 (m, 1H, CH), 3.71 (m, 2H, CH₂), 3.52 (m, 2H, 2×CH), 3.20 (m,1H, CH), 2.59 (m, 2H, CH₂), 1.59 (m, 2H, CH₂), 1.31 (m, 4H, CH₂), 0.93(m, 3H, CH₃) ppm.

Example 66(3R,4R,5S,6R)-3-[4-(3-Chloropropyl)triazol-1-yl]-6-(hydroxymethyl)tetrahydropyran-2,4,5-triol

Example 66 was synthesized from 2-azido-2-deoxyglucose and5-chloro-pent-1-yne following the CuAAC procedure described in synthesismethod E.

Yield: 36 mg (117 μmol, 80.0%), white solid.

TLC: R_(f)=0.286 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=308.21 [M+H]⁺; calculated: 308.09; t_(R)1 (λ=220nm): 0.72 min; t_(R)2 (λ=220 nm): 0.74 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.75 (s, 1H, NCH), 6.32 (d, J=4.4 Hz, 1H,OH), 5.24 (d, J=5.6 Hz, 1H, OH), 4.91 (dd, J=4.1 Hz, 1H, CH), 4.79 (d,J=5.0 Hz, 1H, OH), 4.63 (dd, J=5.9 Hz, 1H, OH), 4.32 (dd, J=8.2 Hz, 1H,OH), 3.92 (m, 1H, CH), 3.68 (t, J=6.5 Hz, 2H, CH₂), 3.43 (m, 3H, 3×CH),3.10 (m, 1H, CH₂), 2.91 (m, 1H, CH₂), 2.00 (m, 4H, 2×CH₂) ppm.

Example 67(3R,4R,5S,6R)-6-(Hydroxymethyl)-3-[4-[7-(4-nitro-2,1,3-benzoxadiazol-7-yl)heptyl]triazol-1-yl]tetrahydropyran-2,4,5-triol

Step 1: 4-nitro-N-oct-7-ynyl-2,1,3-benzoxadiazol-7-amine

To a solution of 300 mg (1.49 mmol) 4-chloro-7-nitrobenzofurazane in 4mL dry dimethylformamide was added 216 mg (1.64 mmol) 7-octyn-1-amineand 527 μl (2.98 mmol) N,N-diisopropylethylamine. The reaction mixturewas stirred at room temperature for 2 days and evaporated.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica40 g; flow rate: 40 mL/min; wavelength for detection: 254 nm; eluent:(A) n-heptane, (B) ethylacetate.

MPLC Gradient

start % B end % B duration [min] 0.0 0.0 1.6 0.0 27.7 16.6 27.7 79.819.9 79.8 0.0 0.0 0.0 0.0 2.9

Yield: 96 mg (0.333 mmol, 22.4%), orange solid.

TLC: R_(f)=0.689 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=289.10 [M+H]⁺; calculated: 289.30; t_(R) (λ=220 nm):1.25 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=9.53 (s, 1H, NH), 8.51 (d, J=8.8 Hz, 2H,ArH), 6.41 (d, J=8.9 Hz, 1H, OH), 5.23 (d, J=5.7 Hz, 1H, OH), 4.87 (dd,J=4.2 Hz, 1H, CH), 4.56 (d, J=6.7 Hz, 1H, OH), 4.63 (dd, J=5.9 Hz, 1H,OH), 4.32 (dd, J=8.2 Hz, 1H, OH), 3.71 (m, 2H, 2×CH), 3.45 (m, 3H,3×CH), 3.31 (m, 1H, CH₂), 3.20 (m, 1H, CH₂), 2.59 (m, 2H, CH₂), 1.65 (m,6H, 3×CH₂), 1.36 (m, 4H, 2×CH₂) ppm.

Step 2:(3R,4R,5S,6R)-6-(hydroxymethyl)-3-[4-[7-(4-nitro-2,1,3-benzoxadiazol-7-yl)heptyl]triazol-1-yl]tetrahydropyran-2,4,5-triol

Example 67 was synthesized from 2-azido-2-deoxyglucose and example 67,step 1 following the CuAAC procedure described in synthesis method E.

Yield: 32 mg (64 μmol, 44.3%), orange solid.

TLC: R_(f)=0.833 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=494.24 [M+H]⁺; calculated: 494.19; t_(R)1 (λ=220nm): 1.33 min; t_(R)2 (λ=220 nm): 1.35 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=9.53 (s, 1H, NH), 8.51 (d, J=8.8 Hz, 2H,ArH), 8.00 (s, 1H, NCH), 6.41 (d, J=8.9 Hz, 1H, OH), 5.23 (d, J=5.7 Hz,1H, OH), 4.87 (dd, J=4.2 Hz, 1H, CH), 4.56 (d, J=6.7 Hz, 1H, OH), 4.63(dd, J=5.9 Hz, 1H, OH), 4.32 (dd, J=8.2 Hz, 1H, OH), 3.71 (m, 2H, 2×CH),3.45 (m, 3H, 3×CH), 3.31 (m, 1H, CH₂), 3.20 (m, 1H, CH₂), 2.59 (m, 2H,CH₂), 1.65 (m, 6H, 3×CH₂), 1.36 (m, 4H, 2×CH₂) ppm.

Example 685-(Dimethylamino)-N-[[1-[[(2R,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methyl]triazol-4-yl]methyl]naphthalene-1-sulfonamide

Example 68 was synthesized from 6-azido-6-deoxyglucose and example 54,step 1 following the CuAAC procedure described in synthesis method E.

Yield: 18 mg (36.47 μmol, 49.9%), white-yellow solid.

TLC: R_(f)=0.045 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=494.25 [M+H]⁺; calculated: 494.16; t_(R)1 (λ=220nm): 1.18 min; t_(R)2 (λ=220 nm): 1.20 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.46 (d, J=8.4 Hz, 2H, ArH), 8.13 (d, J=7.0Hz, 1H, ArH), 7.84 (s, 1H, NCH), 7.67 (s, 1H, SNH), 7.57 (m, 2H, ArH),7.25 (d, J=7.2 Hz, 1H, ArH), 6.31 (d, J=6.4 Hz, 1H, OH), 5.23 (d, J=6.3Hz, 1H, OH), 4.92 (d, J=6.2 Hz, 1H, OH), 4.85 (dd, J=6.4 Hz, 1H, CH),4.58 (d, J=6.0 Hz, 1H, OH), 6=4.21 (m, 1H, CH₂), 4.07 (s, 2H, NCH₂),3.89 (m, 1H, CH₂), 3.47 (m, 1H, CH), 3.14 (m, 1H, CH), 2.96 (m, 1H, CH),2.81 (s, 6H, 2×NCH₃) ppm.

Example 69(3R,4S,5S,6R)-6-[(4-Phenyltriazol-1-yl)methyl]tetrahydropyran-2,3,4,5-tetrol

Example 69 was synthesized from 6-azido-6-deoxyglucose andethynylbenzene following the CuAAC procedure described in synthesismethod E.

Yield: 36 mg (117 μmol, 80.1%), white solid.

TLC: R_(f)=0.242 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=308.16 [M+H]⁺; calculated: 308.12; t_(R)1 (λ=220nm): 0.90 min; t_(R)2 (λ=220 nm): 0.97 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.49 (s, 1H, NCH), 7.86 (dd, J=1.3 Hz, 2H,ArH), 7.46 (m, 2H, ArH), 7.42 (m, 1H, ArH), 6.36 (d, J=6.0 Hz, 1H, OH),5.31 (d, J=5.8 Hz, 1H, OH), 4.96 (t, J=5.7 Hz, 1H, CH), 4.90 (d, J=4.2Hz, 1H, OH), 4.65 (d, J=6.1 Hz, 1H, OH), 4.41 (m, 2H, CH₂), 3.99 (m, 1H,CH), 3.49 (m, 1H, CH), 3.22 (m, 1H, CH), 3.00 (m, 1H, CH) ppm.

¹³C-NMR (150 MHz, DMSO-d₆): δ=146.03 (s, C), 130.84 (s, C), 128.85 (s,CH), 127.75 (s, CH), 125.09 (s, CH), 122.25 (s, CH), 92.35 (s, CH),72.69 (s, CH), 72.07 (s, CH), 71.89 (s, CH), 69.97 (s, CH), 61.26 (s,CH₂) ppm.

Example 70(3R,4S,5S,6R)-6-[[4-(p-Tolyl)triazol-1-yl]methyl]tetrahydropyran-2,3,4,5-tetrol

Example 70 was synthesized from 6-azido-6-deoxyglucose and1-ethynyl-4-methylbenzene following the CuAAC procedure described insynthesis method E.

Yield: 32 mg (100 μmol, 68.1%), white solid.

TLC: R_(f)=0.250 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=322.19 [M+H]⁺; calculated: 322.13; t_(R)1 (λ=220nm): 1.12 min; t_(R)2 (λ=220 nm): 1.16 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.49 (s, 1H, NCH), 7.72 (d, J=8.0 Hz, 2H,ArH), 7.21 (d, J=8.0 Hz, 2H, ArH), 6.43 (d, J=4.4 Hz, 1H, OH), 5.30 (d,J=5.4 Hz, 1H, OH), 4.94 (d, J=5.4 Hz, 1H, OH), 4.90 (dd, J=4.2 Hz, 1H,CH), 4.58 (d, J=5.3 Hz, 1H, OH), 4.40 (m, 1H, CH₂), 4.00 (m, 1H, CH₂),3.49 (m, 1H, CH), 3.17 (m, 1H, CH), 3.06 (m, 1H, CH), 2.94 (m, 1H, CH),2.36 (s, 3H, CH₃) ppm.

Example 71(3R,4S,5S,6R)-6-[[4-(3-Phenylpropyl)triazol-1-yl]methyl]tetrahydropyran-2,3,4,5-tetrol

Example 71 was synthesized from 6-azido-6-deoxyglucose andpent-4-yn-1-ylbenzene following the CuAAC procedure described insynthesis method E.

Yield: 43 mg (123 μmol, 84.2%), white solid.

TLC: R_(f)=0.273 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=350.18 [M+H]⁺; calculated: 350.16; t_(R)1 (λ=220nm): 1.29 min; t_(R)2 (λ=220 nm): 1.35 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.72 (s, 1H, NCH), 7.25 (m, 2H, ArH), 7.20(m, 3H, ArH), 6.31 (d, J=5.0 Hz, 1H, OH), 5.23 (d, J=5.6 Hz, 1H, OH),4.89 (d, J=6.5 Hz, 1H, CH), 4.80 (d, J=5.0 Hz, 1H, OH), 4.55 (dd, J=5.5Hz, 1H, OH), 4.31 (m, 1H, CH₂), 3.93 (m, 1H, CH₂), 3.49 (m, 2H, 2×CH),3.11 (m, 1H, CH), 2.97 (m, 1H, CH), 2.63 (m, 4H, CH₂), 1.90 (m, 2H, CH₂)ppm.

Example 72Methyl-4-[1-[[(2R,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methyl]triazol-4-yl]-benzoate

Example 72 was synthesized from 6-azido-6-deoxyglucose and4-ethynylmethylbenzoate following the CuAAC procedure described insynthesis method E.

Yield: 34 mg (93 μmol, 63.6%), white solid.

TLC: R_(f)=0.258 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=366.23 [M+H]⁺; calculated: 366.12; t_(R)1 (λ=220nm): 1.05 min; t_(R)2 (λ=220 nm): 1.08 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.59 (s, 1H, NCH), 8.01 (m, 4H, ArH), 6.37(d, J=5.0 Hz, 1H, OH), 5.31 (d, J=5.6 Hz, 1H, OH), 4.94 (d, J=4.8 Hz,1H, OH), 4.89 (dd, J=4.3 Hz, 1H, CH), 4.61 (d, J=5.1 Hz, 1H, OH), 4.44(m, 1H, CH₂), 4.02 (m, 1H, CH₂), 3.90 (s, 3H, OCH₃), 3.51 (m, 1H, CH),3.19 (m, 1H, CH), 3.08 (m, 1H, CH), 2.91 (m, 1H, CH) ppm.

Example 73(3R,4S,5S,6R)-6-[[4-[[Benzyl(methyl)amino]methyl]triazol-1-yl]methyl]tetrahydropyran-2,3,4,5-tetrol

Example 73 was synthesized from 6-azido-6-deoxyglucose andN-benzyl-N-methylprop-2-yn-1-amine following the CuAAC proceduredescribed in synthesis method E.

Yield: 36 mg (99 μmol, 67.6%), white solid.

TLC: R_(f)=0.053 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=365.20 [M+H]⁺; calculated: 365.18; t_(R) (ELSD):0.37 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.85 (s, 1H, NCH), 7.31 (m, 5H, ArH), 6.33(d, J=6.1 Hz, 1H, OH), 5.24 (d, J=5.8 Hz, 1H, OH), 4.90 (d, J=5.6 Hz,1H, OH), 4.87 (dd, J=6.0 Hz, 1H, CH), 4.58 (d, J=5.0 Hz, 1H, OH), 4.41(m, 1H, CH₂), 3.97 (m, 1H, CH₂), 3.62 (s, 3H, NCH₃), 3.50 (m, 2H, 2×CH),3.15 (m, 1H, CH), 2.96 (m, 1H, CH), 2.10 (m, 4H, 2×NCH₂) ppm.

Example 74(3R,4S,5S,6R)-6-[[4-(6-Methoxy-2-naphthyl)triazol-1-yl]methyl]tetrahydropyran-2,3,4,5-tetrol

Example 74 was synthesized from 6-azido-6-deoxyglucose and2-ethynyl-6-methoxynaphthalene following the CuAAC procedure describedin synthesis method E.

Yield: 43 mg (111 μmol, 75.9%), white solid.

TLC: R_(f)=0.288 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=388.20 [M+H]⁺; calculated: 388.14; t_(R)1 (λ=220nm): 1.30 min; t_(R)2 (λ=220 nm): 1.33 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.51 (s, 1H, NCH), 7.86 (m, 4H, ArH), 7.34(s, 2H, ArH), 6.39 (d, J=5.8 Hz, 1H, OH), 5.41 (d, J=6.1 Hz, 1H, OH),4.94 (d, J=6.0 Hz, 1H, OH), 4.90 (dd, J=6.3 Hz, 1H, CH), 4.58 (d, J=5.2Hz, 1H, OH), 4.42 (m, 1H, CH₂), 4.01 (m, 1H, CH₂), 3.90 (s, 3H, OCH₃),3.51 (m, 1H, CH), 3.19 (m, 1H, CH), 3.06 (m, 1H, CH), 2.97 (m, 1H, CH)ppm.

Example 75(3R,4S,5S,6R)-6-[[4-[[(4-Nitro-2,1,3-benzoxadiazol-7-yl)amino]methyl]triazol-1-yl]methyl]tetrahydropyran-2,3,4,5-tetrol

Example 75 was synthesized from 6-azido-6-deoxyglucose and example 63,step 1 following the CuAAC procedure described in synthesis method E.

Yield: 56 mg (132 μmol, 90.5%), orange solid.

TLC: R_(f)=0.121 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=424.16 [M+H]⁺; calculated: 424.11; t_(R)1 (λ=220nm): 0.83 min; t_(R)2 (λ=220 nm): 0.87 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=9.85 (s, 1H, NH) 8.53 (d, J=8.4 Hz, 1H,ArH), 8.01 (s, 1H, NCH), 6.51 (d, J=8.4 Hz, 1H, ArH), 6.33 (d, J=6.0 Hz,1H, OH), 5.24 (d, J=6.1 Hz, 1H, OH), 4.94 (d, J=6.2 Hz, 1H, OH), 4.86(dd, J=5.8 Hz, 1H, CH), 4.65 (s, 2H, NCH₂), 4.57 (dd, J=6.0 Hz, 1H, OH),4.37 (m, 1H, CH₂), 3.91 (m, 1H, CH₂), 3.49 (m, 1H, CH), 3.14 (m, 1H,CH), 2.95 (m, 1H, CH) ppm.

Example 76(3R,4S,5S,6R)-6-[[4-(Cyclohexylmethyl)triazol-1-yl]methyl]tetrahydropyran-2,3,4,5-tetrol

Example 76 was synthesized from 6-azido-6-deoxyglucose andprop-2-yn-1-ylcyclohexane following the CuAAC procedure described insynthesis method E.

Yield: 33 mg (101 μmol, 68.9%), white-yellow solid.

TLC: R_(f)=0.220 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=328.24 [M+H]⁺; calculated: 328.18; t_(R)1 (λ=220nm): 1.23 min; t_(R)2 (λ=220 nm): 1.29 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.68 (s, 1H, NCH), 6.33 (d, J=5.0 Hz, 1H,OH), 5.22 (d, J=5.5 Hz, 1H, OH), 4.87 (dd, J=4.3 Hz, 1H, CH), 4.78 (d,J=5.0 Hz, 1H, OH), 4.59 (dd, J=2.3 Hz, 1H, OH), 4.55 (d, J=6.7 Hz, 1H,OH), 4.36 (m, 1H, CH₂), 3.92 (m, 1H, CH₂), 3.44 (m, 1H, CH), 3.12 (m,1H, CH), 2.90 (m, 1H, CH), 2.49 (m, 2H, CH₂), 1.64 (m, 6H, 3×CH₂), 1.53(m, 1H, CH), 1.17 (m, 4H, 2×CH₂), 0.95 (m, 2H, CH₂) ppm.

Example 77(3R,4S,5S,6R)-6-[(4-Pentyltriazol-1-yl)methyl]tetrahydropyran-2,3,4,5-tetrol

Example 77 was synthesized from 6-azido-6-deoxyglucose and hept-1-ynefollowing the CuAAC procedure described in synthesis method E.

Yield: 7 mg (23 μmol, 15.9%), white-yellow solid.

TLC: R_(f)=0.235 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=302.25 [M+H]⁺; calculated: 302.16; t_(R)1 (λ=220nm): 1.12 min; t_(R)2 (λ=220 nm): 1.17 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.70 (s, 1H, NCH), 6.33 (d, J=4.8 Hz, 1H,OH), 5.23 (d, J=5.7 Hz, 1H, OH), 4.87 (dd, J=4.0 Hz, 1H, CH), 4.78 (d,J=4.9 Hz, 1H, OH), 4.60 (dd, J=2.3 Hz, 1H, OH), 4.55 (d, J=6.6 Hz, 1H,OH), 4.31 (m, 1H, CH₂), 3.92 (m, 1H, CH₂), 3.52 (m, 1H, CH), 3.12 (m,1H, CH), 2.94 (m, 1H, CH), 2.58 (t, J=7.5 Hz, 2H, CH₂), 1.58 (m, 2H,CH₂), 1.30 (m, 4H, CH₂), 0.87 (m, 3H, CH₃) ppm.

Example 78(3R,4S,5S,6R)-6-[[4-(3-Chloropropyl)triazol-1-yl]methyl]tetrahydropyran-2,3,4,5-tetrol

Example 78 was synthesized from 6-azido-6-deoxyglucose and5-chloro-pent-1-yne following the CuAAC procedure described in synthesismethod E.

Yield: 24 mg (78 μmol, 53.3%), white-yellow solid.

TLC: R_(f)=0.182 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=308.17 [M+H]⁺; calculated: 308.09; t_(R)1 (λ=220nm): 0.70 min; t_(R)2 (λ=220 nm): 0.72 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.78 (s, 1H, NCH), 6.33 (d, J=5.0 Hz, 1H,OH), 5.24 (d, J=5.7 Hz, 1H, OH), 4.89 (dd, J=4.9 Hz, 1H, CH), 4.79 (d,J=5.3 Hz, 1H, OH), 4.61 (dd, J=2.2 Hz, 1H, OH), 4.55 (d, J=6.6 Hz, 1H,OH), 4.34 (m, 1H, CH₂), 3.93 (m, 1H, CH₂), 3.68 (t, J=6.6 Hz, 2H, CH₂),3.44 (m, 2H, 2×CH), 3.15 (m, 1H, CH), 2.91 (m, 1H, CH), 2.07 (m, 4H,2×CH₂) ppm.

Example 79(3R,4S,5S,6R)-6-[[4-[6-[(4-Nitro-2,1,3-benzoxadiazol-7-yl)amino]hexyl]triazol-1-yl]methyl]tetrahydropyran-2,3,4,5-tetrol

Example 79 was synthesized from 6-azido-6-deoxyglucose and example 67,step 1 following the CuAAC procedure described in synthesis method E.

Yield: 20 mg (40.53 μmol, 55.4%), orange oil.

TLC: R_(f)=0.222 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=494.23 [M+H]⁺; calculated: 494.19; t_(R)1 (λ=220nm): 1.33 min; t_(R)2 (λ=220 nm): 1.35 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=9.50 (s, 1H, NH), 8.49 (d, J=8.8 Hz, 2H,ArH), 769 (s, 1H, NCH), 6.41 (d, J=5.9 Hz, 1H, OH), 5.23 (d, J=5.7 Hz,1H, OH), 4.91 (d, J=6.7 Hz, 1H, OH), 4.87 (dd, J=4.0 Hz, 1H, CH), 4.63(dd, J=5.9 Hz, 1H, OH), 4.56 (d, J=6.2 Hz, 1H, OH), 4.30 (m, 1H, CH₂),3.91 (m, 1H, CH₂), 3.49 (m, 2H, 2×CH), 3.17 (m, 1H, CH), 2.94 (m, 1H,CH), 2.58 (m, 2H, CH₂), 1.65 (m, 4H, 2×CH₂), 1.40 (m, 6H, 3×CH₂) ppm.

Example 805-(Dimethylamino)-N-[[1-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]triazol-4-yl]methyl]naphthalene-1-sulfonamide

Example 80 was synthesized from 1-azido-1-deoxyglucose and example 54,step 1 following the CuAAC procedure described in synthesis method E.

Yield: 46 mg (94 μmol, 63.7%), white-yellow solid.

TLC: R_(f)=0.129 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=494.26 [M+H]⁺; calculated: 494.16; t_(R) (λ=220 nm):1.20 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.48 (d, J=8.4 Hz, 1H, ArH), 8.32 (d, J=8.6Hz, 1H, ArH), 8.14 (dd, J=1.1 Hz, 1H, ArH), 8.02 (s, 1H, NCH), 7.62 (m,2H, ArH), 7.27 (d, J=7.6 Hz, 1H, ArH), 5.49 (d, J=9.2 Hz, 1H, OH), 5.31(d, J=6.2 Hz, 1H, OH), 5.23 (d, J=4.9 Hz, 1H, OH), 5.13 (d, J=5.4 Hz,1H, CH), 4.62 (dd, J=5.4 Hz, 1H, OH), 4.08 (d, J=5.1 Hz, 1H, NCH₂), 3.68(m, 2H, CH₂), 3.43 (m, 2H, 2×CH), 3.19 (m, 1H, CH), 2.84 (s, 6H, 2×NCH₃)ppm.

Example 81(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-(4-phenyltriazol-1-yl)tetrahydropyran-3,4,5-triol

Example 81 was synthesized from 1-azido-1-deoxyglucose andethynylbenzene following the CuAAC procedure described in synthesismethod E.

Yield: 40 mg (130 μmol, 89.0%), white solid.

TLC: R_(f)=0.295 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=308.14 [M+H]⁺; calculated: 308.12; t_(R) (λ=220 nm):0.92 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.81 (s, 1H, NCH), 7.88 (d, J=8.0 Hz, 2H,ArH), 7.46 (t, J=7.5 Hz, 2H, ArH), 7.34 (d, J=7.5 Hz, 1H, ArH), 5.57 (d,J=9.2 Hz, 1H, CH), 5.42 (d, J=5.8 Hz, 1H, OH), 5.31 (d, J=4.9 Hz, 1H,OH), 5.16 (d, J=5.5 Hz, 1H, OH), 4.62 (dd, J=5.6 Hz, 1H, OH), 3.81 (m,1H, CH₂), 3.73 (m, 1H, CH₂), 3.49 (m, 2H, 2×CH), 3.17 (m, 1H, CH₂) ppm.

¹³C-NMR (150 MHz, DMSO-d₆): δ=146.31 (s, C), 130.62 (s, C), 128.91 (s,CH), 127.92 (s, CH), 125.15 (s, CH), 120.47 (s, CH), 87.68 (s, CH),79.95 (s, CH), 76.85 (s, CH), 72.19 (s, CH), 69.60 (s, CH), 60.76 (s,CH₂) ppm.

Example 82(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-[4-(p-tolyl)triazol-1-yl]tetrahydropyran-3,4,5-triol

Example 82 was synthesized from 1-azido-1-deoxyglucose and1-ethynyl-4-methylbenzene following the CuAAC procedure described insynthesis method E.

Yield: 39 mg (121 μmol, 83.0%), white solid.

TLC: R_(f)=0.326 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=322.20 [M+H]⁺; calculated: 322.13; t_(R) (λ=220 nm):1.14 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.76 (s, 1H, NCH), 7.77 (d, J=8.3 Hz, 2H,ArH), 7.27 (d, J=8.3 Hz, 2H, ArH), 5.55 (d, J=9.1 Hz, 1H, OH), 5.41 (d,J=5.9 Hz, 1H, OH), 5.30 (d, J=5.1 Hz, 1H, OH), 5.15 (d, J=5.5 Hz, 1H,CH), 4.61 (dd, J=5.5 Hz, 1H, OH), 3.80 (m, 1H, CH₂), 3.72 (m, 1H, CH₂),3.45 (m, 2H, 2×CH), 3.26 (m, 1H, CH), 2.34 (s, 3H, CH₃) ppm.

Example 83(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-[4-(3-phenylpropyl)triazol-1-yl]tetrahydropyran-3,4,5-triol

Example 83 was synthesized from 1-azido-1-deoxyglucose andpent-4yn-lylbenzene following the CuAAC procedure described in synthesismethod E.

Yield: 50 mg (143 μmol, 97.9%), white solid.

TLC: R_(f)=0.356 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=350.21 [M+H]⁺; calculated: 350.16; t_(R) (λ=220 nm):1.27 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.07 (s, 1H, NCH), 7.25 (m, 5H, ArH), 5.46(d, J=9.2 Hz, 1H, OH), 5.31 (d, J=5.9 Hz, 1H, OH), 5.24 (d, J=5.0 Hz,1H, OH), 5.12 (d, J=5.5 Hz, 1H, CH), 4.59 (dd, J=5.0 Hz, 1H, OH), 3.72(m, 2H, CH₂), 3.42 (m, 2H, 2×CH), 3.21 (m, 1H, CH), 2.63 (m, 4H, 2×CH₂),1.90 (m, 2H, CH₂) ppm.

Example 84Methyl-4-[1-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]triazol-4-yl]benzoate

Example 84 was synthesized from 1-azido-1-deoxyglucose and4-ethynylmethylbenzoate following the CuAAC procedure described insynthesis method E.

Yield: 44 mg (120 μmol, 82.4%), white solid.

TLC: R_(f)=0.062 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=366.12 [M+H]⁺; calculated: 366.12; t_(R)1 (λ=220nm): 0.97 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=9.00 (s, 1H, NCH), 8.05 (s, 4H, ArH), 5.60(d, J=9.3 Hz, 1H, OH), 5.44 (d, J=5.9 Hz, 1H, OH), 5.32 (d, J=4.8 Hz,1H, OH), 5.17 (d, J=5.5 Hz, 1H, CH), 4.62 (dd, J=5.8 Hz, 1H, OH), 3.87(s, 3H, OCH₃), 3.87 (m, 1H, CH₂), 3.80 (m, 1H, CH₂), 3.49 (m, 1H, CH),3.43 (m, 1H, CH), 3.27 (m, 1H, CH) ppm.

Example 85(2R,3R,4S,5S,6R)-2-[4-[[Benzyl(methyl)amino]methyl]triazol-1-yl]-6-(hydroxymethyl)tetrahydropyran-3,4,5-triol

Example 85 was synthesized from 1-azido-1-deoxyglucose andN-benzyl-N-methylprop-2-yn-1-amine following the CuAAC proceduredescribed in synthesis method E.

Yield: 51 mg (140 μmol, 95.7%), white solid.

TLC: R_(f)=0.091 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=365.20 [M+H]⁺; calculated: 365.17; t_(R) (ELSD):0.34 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.19 (s, 1H, NCH), 7.33 (m, 4H, ArH), 7.25(m, 1H, ArH), 5.50 (d, J=9.4 Hz, 1H, OH), 5.34 (d, J=6.1 Hz, 1H, OH),5.24 (d, J=5.0 Hz, 1H, OH), 5.12 (d, J=5.7 Hz, 1H, CH), 4.60 (dd, J=5.6Hz, 1H, OH), 3.77 (m, 1H, CH₂), 3.70 (m, 1H, CH₂), 3.62 (s, 2H, NCH₂),3.52 (s, 2H, NCH₂), 3.43 (m, 1H, CH), 3.38 (m, 1H, CH), 3.24 (m, 1H, CH)2.11 (s, 3H, NCH₃) ppm.

Example 86(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-[4-(6-methoxy-2-naphthyl)triazol-1-yl]tetrahydropyran-3,4,5-triol

Example 86 was synthesized from 1-azido-1-deoxyglucose and2-ethynyl-6-methoxynaphthalene following the CuAAC procedure describedin synthesis method E.

Yield: 42 mg (108 μmol, 74.1%), white solid.

TLC: R_(f)=0.318 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=388.10 [M+H]⁺; calculated: 388.14; t_(R) (λ=220 nm):0.95 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.51 (s, 1H, NCH), 7.86 (m, 4H, ArH), 7.34(s, 2H, ArH), 6.39 (d, J=5.8 Hz, 1H, OH), 5.41 (d, J=6.1 Hz, 1H, OH),4.94 (d, J=6.0 Hz, 1H, OH), 4.90 (dd, J=6.3 Hz, 1H, CH), 4.58 (d, J=5.2Hz, 1H, OH), 4.42 (m, 1H, CH₂), 4.01 (m, 1H, CH₂), 3.90 (s, 3H, OCH₃),3.51 (m, 1H, CH), 3.19 (m, 1H, CH), 3.06 (m, 1H, CH), 2.97 (m, 1H, CH)ppm.

Example 87Ethyl-2-diethoxyphosphoryl-3-[1-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-pyran-2-yl]triazol-4-yl]propanoate

Example 87 was synthesized from 1-azido-1-deoxyglucose and2-(diethoxyphosphoryl)ethylpent-4-ynoate following the CuAAC proceduredescribed in synthesis method E.

Yield: 48 mg (103 μmol, 70.2%), white solid.

TLC: R_(f)=0.258 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=468.26 [M+H]⁺; calculated: 468.17; t_(R) (λ=220 nm):1.04 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.05 (s, 1H, NCH), 5.46 (d, J=9.1 Hz, 1H,OH), 5.30 (d, J=6.3 Hz, 1H, OH), 5.24 (d, J=4.7 Hz, 1H, OH), 5.12 (d,J=5.5 Hz, 1H, CH), 4.59 (dd, J=5.3 Hz, 1H, OH), 4.08 (m, 2H, CH₂), 3.70(m, 2H, CH₂), 3.43 (m, 4H, 4×CH), 3.00 (m, 1H, CH), 1.25 (m, 6H, 3×CH₂),1.13 (s, 9H, 3×CH₃) ppm.

Example 88(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-[4-[[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]methyl]triazol-1-yl]tetrahydropyran-3,4,5-triol

Example 88 was synthesized from 1-azido-1-deoxyglucose and example 63,step 1 following the CuAAC procedure described in synthesis method E.

Yield: 58 mg (138 μmol, 93.7%).

TLC: R_(f)=0.152 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=424.13 [M+H]⁺; calculated: 424.16; t_(R)1 (λ=220nm): 0.84 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=9.87 (s, 1H, NH), 8.51 (d, J=8.9 Hz, 1H,ArH), 8.35 (s, 1H, NCH), 6.55 (d, J=8.9 Hz, 1H, ArH), 5.50 (d, J=9.3 Hz,1H, OH), 5.32 (d, J=6.0 Hz, 1H, OH), 5.25 (d, J=4.9 Hz, 1H, OH), 5.12(d, J=5.5 Hz, 1H, CH), 4.80 (s, 2H, NCH₂), 4.60 (dd, J=5.5 Hz, 1H, OH),3.70 (m, 2H, CH₂), 3.43 (m, 2H, 2×CH), 3.19 (m, 1H, CH) ppm.

Example 89(2R,3R,4S,5S,6R)-2-[4-(Cyclohexylmethyl)triazol-1-yl]-6-(hydroxymethyl)tetrahydropyran-3,4,5-triol

Example 89 was synthesized from 1-azido-1-deoxyglucose andprop-2-yn-1-ylcyclohexane following the CuAAC procedure described insynthesis method E.

Yield: 35 mg (107 μmol, 73.1%), white solid.

TLC: R_(f)=0.258 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=328.26 [M+H]⁺; calculated: 328.18; t_(R) (λ=220 nm):1.27 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.99 (s, 1H, NCH), 5.45 (d, J=9.3 Hz, 1H,OH), 5.30 (d, J=6.0 Hz, 1H, OH), 5.23 (d, J=5.0 Hz, 1H, OH), 5.11 (d,J=5.3 Hz, 1H, CH), 4.59 (dd, J=5.5 Hz, 1H, OH), 3.71 (m, 2H, CH₂), 3.42(m, 2H, 2×CH), 3.22 (m, 2H, 2×CH), 1.66 (m, 6H, 3×CH₂), 1.54 (m, 1H,CH), 1.17 (m, 4H, 2×CH₂), 0.95 (m, 2H, CH₂) ppm.

Example 90(2R,3R,4S,5S,6R)-2-[4-(3-Chloropropyl)triazol-1-yl]-6-(hydroxymethyl)tetrahydropyran-3,4,5-triol

Example 90 was synthesized from 1-azido-1-deoxyglucose and5-chloro-pent-1-yne following the CuAAC procedure described in synthesismethod E.

Yield: 17 mg (55 μmol, 37.8%), white solid.

TLC: R_(f)=0.288 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=308.12 [M+H]⁺; calculated: 308.09; t_(R)1 (λ=220nm): 0.62 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.09 (s, 1H, NCH), 5.46 (d, J=9.1 Hz, 1H,OH), 5.31 (d, J=6.0 Hz, 1H, OH), 5.25 (d, J=4.7 Hz, 1H, OH), 5.12 (d,J=5.8 Hz, 1H, CH), 4.60 (dd, J=5.2 Hz, 1H, OH), 3.74 (m, 2H, CH₂), 3.70(m, 2H, CH₂), 3.39 (m, 2H, 2×CH), 3.22 (m, 2H, 2×CH), 2.77 (m, 2H, CH₂),2.06 (m, 2H, CH₂) ppm.

Example 91[6-(Diethylamino)-9-[2-[[1-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]triazol-4-yl]methylcarbamoyl]phenyl]xanthen-3-ylidene]-diethyl-ammoniumchloride

Step 1:[6-(diethylamino)-9-[2-(prop-2-ynylcarbamoyl)phenyl]xanthen-3-ylidene]-diethyl-ammoniumchloride

To a solution of 500 mg (244 μmol) Rhodamin B in 5 mL dimethylformamidewere added 78 mg (1.39 mmol; 1.4 eq.) propargylamine, 554 mg (1.98 mmol;2 eq.) 2-bromo-1-ethylpyridinium tetrafluoroborate, and 348 μl (1.98mmol; 2 eq.) N,N-diisopropylethylamine. The reaction mixture was stirredat room temperature for 4 hours and evaporated.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica24 g Gold; flow rate: 40 mL/min; wavelength for detection: 254 nm;eluent: (A) dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0.0 0.0 5.0 0.0 100.0 45.0

Yield: 223 mg (0.432 mmol, 43.6%), light pink solid.

TLC: R_(f)=0.622 (dichloromethane/ethanol, 19:1).

LC/MS (ES-API): m/z=480.28 [M+H]⁺; calculated: 480.27; t_(R) (λ=220 nm):2.08 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.80 (m, 1H, ArH), 7.51 (m, 1H, ArH), 7.03(m, 1H, ArH), 6.35 (m, 6H, ArH), 3.79 (d, J=6.9 Hz, 2H, NCH₂), 3.31 (m,8H, H), 2.65 (m, 8H, CH₂), 1.08 (t, J=6.9 Hz, 12H, CH₃) ppm.

Step 2:[6-(diethylamino)-9-[2-[[1-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]triazol-4-yl]methylcarbamoyl]phenyl]xanthen-3-ylidene]-diethyl-ammoniumchloride

Example 91 was synthesized from 1-azido-1-deoxyglucose and example 91,step 1 following the CuAAC procedure described in synthesis method E.

Yield: 33 mg (67 μmol, 46.7%), white-yellow solid.

TLC: R_(f)=0.485 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=686.38 [M+H]⁺; calculated: 686.33; t_(R) (λ=220 nm):1.67 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.09 (s, 1H, NCH), 7.82 (m, 1H, ArH), 7.51(m, 1H, ArH), 7.08 (m, 1H, ArH), 6.33 (m, 6H, ArH), 5.40 (d, J=9.1 Hz,1H, OH), 5.24 (d, J=6.0 Hz, 1H, OH), 5.19 (d, J=4.7 Hz, 1H, OH), 5.08(d, J=5.6 Hz, 1H, CH), 4.63 (dd, J=5.0 Hz, 1H, OH), 3.82 (d, J=6.9 Hz,2H, NCH₂), 3.77 (m, 2H, CH₂), 3.69 (m, 2H, CH₂), 3.42 (m, 2H, 2×CH),3.29 (m, 8H, H), 3.18 (m, 2H, 2×CH), 2.73 (m, 2H, CH₂), 2.60 (m, 8H,CH₂), 2.03 (m, 2H, CH₂), 1.05 (t, J=6.9 Hz, 12H, CH₃) ppm.

Example 92(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-[4-[6-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]hexyl]triazol-1-yl]tetrahydropyran-3,4,5-triol

Example 92 was synthesized from 1-azido-1-deoxyglucose and example 67,step 1 following the CuAAC procedure described in synthesis method E.

Yield: 24 mg (48 μmol, 66.6%), orange solid.

TLC: R_(f)=0.310 (dichloromethane/ethanol, 4:1).

LC/MS (ES-API): m/z=494.25 [M+H]⁺; calculated: 494.19; t_(R) (λ=220 nm):1.34 min (LC/MS—Method 1).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.48 (d, J=8.6 Hz, 1H, ArH), 8.00 (s, 1H,NCH), 8.14 (d, J=8.6 Hz, 1H, ArH), 5.45 (d, J=9.3 Hz, 1H, OH), 5.29 (d,J=6.1 Hz, 1H, OH), 5.24 (d, J=4.9 Hz, 1H, OH), 5.11 (d, J=5.6 Hz, 1H,CH), 4.58 (dd, J=5.5 Hz, 1H, OH), 3.93 (s, 1H, NCH₂), 3.72 (m, 2H, CH₂),3.41 (m, 3H, 3×CH), 3.20 (m, 1H, CH), 1.66 (m, 6H, 3×CH₂), 1.41 (m, 4H,2×CH₂) ppm.

Example 93[(2S,3R,4S,6S)-2,3-Diacetoxy-6-(4-phenylpiperazine-1-carbonyl)tetrahydropyran-4-yl]acetate

A suspension of 7 mg (0.1 eq.) Pd/C (10%) in 3 mL dry methanol (argonatmosphere) was overlayed with hydrogen and stirred at room temperaturefor 30 minutes. A solution of 30 mg example 41, step 1 in 2 mL drymethanol was added. The reaction mixture was stirred at room temperaturefor 3 hours and filtered over celite.

Purification: MPLC CombiFlash Rf (Teledyne ISCO); column: RediSep Silica4 g; flow rate: 12 mL/min; wavelength for detection: 220 nm; eluent: (A)dichloromethane, (B) ethanol.

MPLC Gradient

start % B end % B duration [min] 0 0 4.0 0 5.0 3.0 5.0 5.0 10.0

Yield: 19 mg (42.37 μmol, 63.0%), orange oil.

LC/MS (ES-API): m/z=449.10 [M+H]⁺; calculated: 449.18; t_(R) (λ=220 nm):0.79 min (LC/MS—Method 1).

¹H-NMR (400 MHz, CDCl₃): δ=7.28 (m, 2H, ArH), 6.92 (m, 3H, ArH), 5.55(d, J=7.9 Hz, 1H, CH), 5.13 (d, J=5.2 Hz, 1H, CH), 5.08 (d, J=7.7 Hz,1H, CH), 4.36 (dd, J=1.7 Hz, J=11.6 Hz 1H, CH), 3.99 (m, 1H, NCH₂), 3.82(m, 1H, NCH₂), 3.54 (m, 2H, NCH₂), 3.28 (m, 2H, NCH₂), 3.03 (m, 2H,NCH₂), 2.32 (ddd, J=2.4 Hz, 1H, CH₂), 2.17 (pseudo-q, J=11.6 Hz, 1H,CH₂), 2.11 (s, 3H, CH₃), 2.06 (s, 6H, 2×CH₃) ppm.

Example 94(4-Phenylpiperazin-1-yl)-[(2S,4S,5R)-4,5,6-trihydroxytetrahydropyran-2-yl]methanone

To a solution of 10 mg example 93 in 1 mL methanol 4 μl of a 5.4 Maqueous sodium methoxide solution were added. The reaction mixture wasagitated at room temperature for 10 minutes and controlled by TLC andLC/MS. The reaction mixture was quenched and evaporated

Yield: 4 mg (12.41 μmol, 55.6%), orange oil.

LC/MS (ES-API): m/z=323.05 [M+H]⁺; calculated: 323.15; t_(R) (ELSD):0.11 min (LC/MS—Method 1).

¹H-NMR (400 MHz, MeOD): δ=7.14 (t, J=8.0 Hz, 2H, ArH), 6.88 (d, J=8.0Hz, 2H, ArH), 6.76 (t, J=7.0 Hz, 1H, ArH), 5.12 (d, J=3.6 Hz, 1H, OH),4.78 (dd, J=2.0 Hz, 1H, OH), 4.42 (d, J=7.6 Hz, 1H, OH), 4.33 (dd, J=2.0Hz, 1H, OH), 3.6-4.0 (m, 4H, 4×CH), 3.4-3.0 (m, 8H, 4×NCH₂), 1.94 (dd,J=4.7 Hz, 1H, CH₂), 1.91 (qi, J=12.2 Hz, 1H, CH₂) ppm.

Example 951-[[(3aR,5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[1,3]dioxolo[4,5-a:4′,5′-c]pyran-5-yl]methyl]-4-(4-benzyloxyphenyl)piperazine

To a solution of(3aR,5S,5aR,8aS,8bR)-2,2,7,7-tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[1,3]dioxolo[4,5-a:4′,5′-c]pyran-5-carbaldehyde(300 mg, 1.16 mmol, 1 eq.), 1-(4-(benzyloxy)phenyl)piperazine (374 mg,1.39 mmol, 1.2 eq.), and acetic acid (132 μl, 2.32 mmol, 2 eq.) inmethanol was added sodium borohydride (147 mg, 2.32 mmol, 2 eq.).

The reaction mixture was agitated overnight at room temperature andevaporated. The residue was separated between ethylacetate and asaturated solution of NaHCO₃. The aqueous phase was extracted twice withethylacetate. The organic phase was dried with MgSO₄, evaporated invacuo, and purified by flash chromatography on silica gel (100% heptanetill 100% ethylacetate in 40 minutes).

Yield: 394 mg (771 μmol, 66%), oil.

LC/MS (ES-API): m/z=511.2 [M+H]+; calculated: 511.6; t_(R) (λ=220 nm):0.77 min (LC/MS—Method 2).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.44-7.28 (m, 5H), 6.86 (s, 4H), 5.45 (d,1H, J=5.1 Hz), 5.01 (s, 2H), 4.57 (dd, 1H, J=7.9, 2.2 Hz), 4.31 (dd, 1H,J=5.1, 2.34 Hz), 4.21 (dd, 1H, J=7.9, 1.6 Hz), 2.98 (t, 4H, J=4.9 Hz),2.65-2.57 (m, 3H), 2.42 (dd, 1H, J=12.7, 7.0 Hz), 1.45 (m, 3H), 1.35 (m,1H), 1.28 (m, 1H) ppm.

Example 96(3R,4S,5R,6R)-6-[[4-(4-Benzyloxyphenyl)piperazin-1-yl]methyl]tetrahydropyran-2,3,4,5-tetrol

Method F

A solution of isopropylidene compounds (1 eq.) in trifluoroaceticacid/water (4/1, v/v) was agitated at room temperature for 2 hours,evaporated under vacuo, and lyophilised.

Example 96 was obtained from example 95 following the isopropylidenedeprotection described in synthesis method F.

Yield: 21 mg (38.4 μmol, quant.).

LC/MS (ES-API): m/z=431.1 [M+H]⁺; calculated: 431.2; t_(R) (λ=220 nm):0.620 min (LC/MS—Method 2).

¹H-NMR (400 MHz, MeOD): δ=7.33 (s, 1H), 7.31 (s, 1H), 7.26 (t, 2H, J=7.1Hz), 7.22-7.17 (m, 1H), 6.87 (q, 4H, J=6.87 Hz), 5.13 (d, 0.9H), 4.93(s, 3H), 4.43 (d, 0.5H, J=6.6 Hz), 4.37 (d, 0.5H, J=9.6 Hz), 3.95 (d,0.7H, J=8.4 Hz), 3.76 (s, 1H), 3.74-3.66 (m, 2H), 3.56-3.38 (m, 4H),3.31 (s, 1H), 3.28 (s, 1H), 3.26 (s, 1H), 3.22 (s, 2H) ppm. Mixture ofdiastereoisomers.

Example 974-[4-[[(3aR,5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[1,3]dioxolo[4,5-a:4′,5′-c]pyran-5-yl]methyl]piperazin-1-yl]phenol

To a solution of example 95 (395 mg, 77 μmol, 1 eq.) in methanol (3 mL)under argon was added Pd/C (8.3 mg, 77 μmol, 0.1 eq.). The reactionmixture was purged with a flux of hydrogen. The reaction mixture wasagitated at room temperature for 1 hour. The reaction mixture wasfiltrated on celite, rinse with methanol, and evaporated in vacuo.

Yield: 366 mg (870 μmol, quant.).

LC/MS (ES-API): m/z=421.1 [M+H]+; calculated: 421.5; t_(R) (λ=220 nm):0.60 min (LC/MS—Method 2).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.72 (d, 2H, J=8.7 Hz), 6.62 (d, 2H, J=8.7Hz), 5.42 (d, 1H, J=5.1 Hz), 4.53 (dd, 1H, J=7.8, 2.3 Hz), 4.26-4.18 (m,2H), 3.93-3.86 (m, 1H), 2.94 (t, 4H, J=5.0 Hz), 2.69-2.49 (m, 4H), 1.50(s, 3H), 1.35 (s, 3H), 1.28 (s, 6H) ppm.

Example 98(2S,3R,4S,5R,6R)-6-[[4-(4-Hydroxyphenyl)piperazin-1-yl]methyl]tetrahydropyran-2,3,4,5-tetrol

Example 98 was obtained from example 97 following the isopropylidenedeprotection described in synthesis method F.

Yield: 31 mg (55 μmol, 93%), oil.

LC/MS (ES-API): m/z=341.2 [M+H]⁺; calculated: 341.2; t_(R)=0.07 min(LC/MS—Method 2).

¹H-NMR (400 MHz, MeOD): δ=6.8 (d, 2H, J=9.0 Hz), 6.64 (d, 2H, J=9.0 Hz),5.12 (d, 1H, J=3.6 Hz), 4.40 (dd, 1H, J=5.8, 1.3 Hz), 4.35 (dd, 1H,J=10.0, 2.5 Hz), 3.95-3.90 (m, 1H), 3.77-3.69 (m, 4H), 3.56-3.46 (m,4H), 3.43-3.40 (m, 2H), 3.31-3.23 (m, 3H) ppm. Mixture ofdiastereoisomers.

Example 991-[[(3aR,5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[1,3]dioxolo[4,5-a:4′,5′-c]pyran-5-yl]methyl]-4-[4-[2-[2-(2-prop-2-ynoxyethoxy)ethoxy]ethoxy]phenyl]piperazine

Step 1: 2-[2-(2-prop-2-ynoxyethoxy)ethoxy]ethyl 4-methylbenzenesulfonate

To a solution of 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethanol (50mg, 266 μmol, 1 eq.) in pyridine (2 mL) were addedN,N,N′,N′-tetramethyl-1,6-diaminohexane (5.8 μl, 27 μmol, 0.1 eq.) and4-methylbenzene-1-sulfonyl chloride (76 mg, 398 μmol, 1.5 eq.). Thereaction mixture was agitated at room temperature for 2 hours. Thereaction mixture was evaporated in vacuo.

Purification: flash chromatography on silica gel (100% heptane till 100%ethylacetate in 30 minutes).

Yield: 62 mg (181 μmol, 68%).

LC/MS (ES-API): m/z=343.1 [M+H]⁺; calculated: 343.4; t_(R) (λ=220 nm):0.804 min (LC/MS—Method 2).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.78 (d, 2H, J=8.5 Hz), 7.48 (d, 2H, J=8.5Hz), 4.15-4.08 (m, 4H), 3.61-3.47 (m, 10H), 3.41 (t, 1H, J=2.5 Hz), 2.42(s, 3H) ppm.

Step 2:1-[[(3aR,5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[1,3]dioxolo[4,5-a:4′,5′-c]pyran-5-yl]methyl]-4-[4-[2-[2-(2-prop-2-ynoxyethoxy)ethoxy]ethoxy]phenyl]piperazine

A solution of example 99, step 1 (41 mg, 120 μmol, 1.2 eq.), example 97(42 mg, 100 μmol, 1 eq.), and Cs₂CO₃ (130 mg, 400 μmol, 4 eq.) indimethylformamide (1 mL) under argon was irradiated in the microwave for10 minutes at 70° C. and 40 minutes at 80° C. The crude mixture wasdiluted with ethylacetate. The organic phase was washed with a saturatedsolution of NaHCO₃, water, dried with MgSO₄, and evaporated in vacuo.

Purification:flash chromatography on silica gel (dichloromethane tilldichloromethane/methanol 9/1 in 30 minutes).

Yield: 16 mg (27 μmol, 27%).

LC/MS (ES-API): m/z=591.3 [M+H]⁺; calculated: 591.3; t_(R) (λ=220 nm):0.70 min (LC/MS—Method 2).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.89-6.81 (m, 4H), 5.57 (d, 1H, J=5.1 Hz),4.60 (dd, 1H, J=7.8, 2.3 Hz), 4.31 (dd, 1H, J=5.1, 2.3 Hz), 4.25-4.19(m, 4H), 4.07 (t, 2H, J=5.1 Hz), 4.02-3.96 (m, 1H), 3.82 (t, 2H, J=5.1Hz), 3.75-3.66 (m, 10H), 3.11 (t, 4H, J=4.5 Hz), 2.80-2.58 (m, 6H),2.43-2.40 (m, 1H), 2.08 (s, 1H), 1.73 (s, 1H), 1.54 (s, 3H), 1.46 (s,3H), 1.34 (s, 3H), 1.33 (s, 3H) ppm.

Example 100(3R,4S,5R,6R)-6-[[4-[4-[2-[2-(2-Prop-2-ynoxyethoxy)ethoxy]ethoxy]phenyl]piperazin-1-yl]methyl]tetrahydropyran-2,3,4,5-tetrol

Example 100 was obtained from example 99 following the isopropylidenedeprotection described in synthesis method F.

Yield: 18 mg (28 μmol, quant).

LC/MS (ES-API): m/z=511.2 [M+H]⁺; calculated: 511.3; t_(R) (λ=220 nm):0.552 min (LC/MS—Method 2).

¹H-NMR (400 MHz, MeOD): δ=6.99 (d, 2H, J=8.9 Hz), 6.91 (d, 2H, J=8.78Hz), 5.23 (d, 0.6H, J=3.5 Hz), 4.52 (d, 0.5H, J=6.0 Hz), 4.47 (d, 0.6H,J=10.0 Hz), 4.23-4.16 (m, 3.2H), 4.11-4.02 (m, 2H), 3.88-3.75 (m, 5H),3.73-3.59 (m, 15H), 3.57-3.49 (m, 1.4H), 3.08 (sl, 2H), 2.84 (s, 1.2H)ppm. Mixture of diastereoisomers.

Example 1011-[[(3aR,5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[1,3]dioxolo[4,5-a:4′,5′-c]pyran-5-yl]met-4-(4-chlorophenyl)piperazine

To a solution of(3aR,5S,5aR,8aS,8bR)-2,2,7,7-tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[1,3]dioxolo[4,5-a:4′,5′-c]pyran-5-carbaldehyde(50 mg, 194 μmol, 1 eq.), 1-(4-chlorophenyl)piperazine (114 mg, 581μmol, 3 eq.), and acetic acid (22 μl, 387 μmol, 2 eq.) in methanol wasadded sodium cyanoboronhydride (25 mg, 387 μmol, 2 eq.). The reactionmixture was agitated overnight at room temperature and evaporated invacuo. The crude mixture was separated between ethylacetate and asaturated solution of NaHCO₃. The aqueous phase was extracted twice withethylacetate. The organic phase was dried with MgSO₄ and evaporated.

Purification: flash chromatography on silica gel (100% heptane till 100%ethylacetate in 40 minutes).

Yield: 63 mg (144 μmol, 74%), oil.

LC/MS (ES-API): m/z=439.2 [M+H]⁺; calculated: 439.9; t_(R) (λ=220 nm):0.713 min (LC/MS—Method 2).

¹H-NMR (400 MHz, DMSO-d₆): δ=7.21 (d, 2H, J=8.9 Hz), 6.93 (d, 2H, J=8.9Hz), 5.45 (d, 1H, J=5.1 Hz), 4.58 (dd, 1H, J=7.9, 2.4 Hz), 4.32 (dd, 1H,J=5.2, 2.3 Hz), 4.21 (dd, 1H, J=7.9, 1.7 Hz), 3.90-3.85 (m, 1H), 3.10(t, 4H, J=4.9 Hz), 2.65-2.57 (m, 4H), 2.43 (dd, 2H, J=13.0, 7.1 Hz),1.45 (s, 3H), 1.35 (s, 3H), 1.28 (s, 6H) ppm.

Example 102(3R,4S,5R,6R)-6-[[4-(4-Chlorophenyl)piperazin-1-yl]methyl]tetrahydropyran-2,3,4,5-tetrol

Example 102 was obtained from example 101 following the isopropylidenedeprotection described in synthesis method F.

Yield: 78 mg (133 μmol, 95%).

LC/MS (ES-API): m/z=359.1 [M+H]⁺; calculated: 359.1; t_(R) (λ=220 nm):0.493 min (LC/MS—Method 2).

¹H-NMR (400 MHz, MeOD): δ=7.27 (d, 2H, J=8.5 Hz), 7.01 (d, 2H, J=8.5Hz). 5.24 (d, 0.5H, J=3.0 Hz), 4.55 (d, 0.5H, J=6.7 Hz), 4.48 (d, 0.5H,J=9.8 Hz), 4.07 (d, 0.5H, J=9.2 Hz), 3.90-3.76 (m, 2H), 3.66-3.30 (m,9H) ppm. Mixture of diastereoisomers.

Example 103N-[2-[2-[2-[[(3aR,5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[1,3]dioxolo[4,5-a:4′,5′-c]pyran-5-yl]methylamino]ethoxy]ethoxy]ethyl]pent-4-ynamide

Step 1: tert-butylN-[2-[2-[2-(pent-4-ynoylamino)ethoxy]ethoxy]ethyl]carbamate

To a solution of pent-4-ynoic acid (43.5 mg, 442 μmol, 1.1 eq.) andtert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (100 mg, 402μmol, 1 eq.) in dimethylformamide (3 mL) was addedN,N-diisopropylethylamine (77 μl, 443 μmol, 1.1 eq.), HOBt (67.8 mg, 443μmol, 1.1 eq.) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (84.9mg, 443 μmol, 1.1 eq.). The reaction mixture was agitated overnight atroom temperature. The crude mixture was diluted with ethylacetate,washed with 0.5 N aqueous HCl, a saturated solution of NaHCO₃, andbrine, dried with MgSO₄, and concentrated in vacuo.

Purification: flash chromatography on silica gel (100% heptane till 100%ethylacetate in 20 minutes, 100% ethylacetate for 10 minutes) Yield: 99mg (301 μmol, 75%).

LC/MS (ES-API): m/z=351.2 [M+Na]+; calculated: 351.4; t_(R): 0.675 min(LC/MS—Method 2).

¹H-NMR (400 MHz, DMSO-d₆): δ=3.49 (s, 4H), 3.41-3.34 (m, 4H), 3.19 (q,2H, J=5.8 Hz), 3.05 (q, 2H, J=5.8 Hz), 2.89 (s, 2H), 2.73 (s, 2H),2.37-2.31 (m, 2H), 2.28-2.24 (m, 2H), 1.90 (s, 1H), 1.37 (s, 9H) ppm.

Step 2: N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]pent-4-ynamide

A solution of example 103, step 1 (98 mg, 298 μmol, 1 eq.) intrifluoroacetic acid/dichloromethane (1/1, v/v, 1 mL) was agitated atroom temperature for 30 minutes. The reaction mixture was evaporatedunder vacuo and lyophilised.

Yield: 108 mg (315 μmol, quant).

LC/MS (ES-API): m/z=229.2 [M+H]⁺; calculated: 229.1; t_(R): 0.123 min(LC/MS—Method 2).

¹H-NMR (400 MHz, MeOD): δ=3.72 (t, 2H, J=4.9 Hz), 3.67 (s, 4H), 3.58 (t,2H, J=5.5 Hz), 3.40 (t, 2H, J=5.5 Hz), 3.15 (t, 2H, J=4.8 Hz), 2.51-2.45(m, 2H), 4.44-2.39 (m, 2H), 2.28 (s, 1H) ppm.

Step 3:N-[2-[2-[2-[[(3aR,5R,5aS,8aS,8bR)-2,2,7,7-tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[1,3]dioxolo[4,5-a:4′,5′-c]pyran-5-yl]methylamino]ethoxy]ethoxy]ethyl]pent-4-ynamide

A solution of(3aR,5S,5aR,8aS,8bR)-2,2,7,7-tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[1,3]dioxolo[4,5-a:4′,5′-c]pyran-5-carbaldehyde(41.5 mg, 160 μmol, 1 eq.), example 103, step 2 (55 mg, 160 μmol, 1eq.), and triethylamine (44.8 μl, 321 μmol, 2 eq.) in methanol (3 mL)containing 4 Å molecular sieves was agitated overnight at roomtemperature. Sodium borohydride (12 mg, 321 μmol, 2 eq.) was added tothe reaction mixture. The reaction mixture was agitated at roomtemperature for 2 hours. A 1 N aqueous solution of sodium hydride wasadded. The reaction mixture was extracted with ether (three times). Theorganic phase was dried with MgSO₄ and evaporated in vacuo.

Purification: flash chromatography on silica gel (dichloromethane tilldichloromethane/methanol 9/1 in 30 minutes).

Yield: 21.4 mg (45.5 μmol, 28%), oil.

LC/MS (ES-API): m/z=471.2 [M+H]⁺; calculated: 471.5; t_(R) (λ=220 nm):0.615 min (LC/MS—Method 2).

¹H-NMR (400 MHz, MeOD): δ=6.70 (t, 1H, J=5.2 Hz), 6.21 (s, 1H), 5.72 (d,1H, J=7.9 Hz), 5.31 (t, 1H, J=9.3 Hz), 5.20 (t, 1H, J=9.3 Hz), 5.11 (t,1H, J=8.5 Hz), 4.07 (d, 1H, J=9.8 Hz), 3.62 (s, 5H), 3.59-3.53 (m, 5H),3.52-3.46 (m, 4H), 3.40-3.31 (m, 1H) ppm.

Example 104N-[2-[2-[2-[[(3R,4S,5R)-3,4,5,6-Tetrahydroxytetrahydropyran-2-yl]methylamino]ethoxy]ethoxy]ethyl]pent-4-ynamide

Example 104 was obtained from example 103 following the isopropylidenedeprotection described in synthesis method F.

Yield: 17.3 mg (44 μmol, quant).

LC/MS (ES-API): m/z=391.1 [M/5+H]⁺; calculated: 391.4; t_(R) (λ=220 nm):0.07 min (LC/MS—Method 2).

Example 105[(2S,3R,4S,5S)-2,3,5-Triacetoxy-6-[2-[2-[2-(pent-4-ynoylamino)ethoxy]ethoxy]ethylcarbamoyl]tetrahydropyran-4-yl]acetate

To a solution of (2R,3S,4S,5R)-2,3,4,5-tetraacetoxycyclohexanecarboxylicacid (101 mg, 280 μmol, 1.2 eq.) in dimethylformamide (3 mL) was addedHATU (106.7 mg, 280 μmol, 1.2 eq.) and 4 Å molecular sieves. After 5minutes example 103, step 2 (80 mg, 233 μmol, 1 eq.) was added. Thereaction mixture was agitated at room temperature for 72 hours. Thereaction mixture was diluted with ethylacetate. The organic phase waswashed with 1N aqueous HCl, saturated NaHCO₃, and brine, dried withMgSO₄, filtered, and evaporated in vacuo.

Purification: flash chromatography on silica gel (dichloromethane tilldichloromethane/methanol 9/1 in 30 minutes)

Yield: 74.45 mg (130 μmol, 56%), oil.

LC/MS (ES-API): m/z=513.2 [M-OAc]+; calculated: 513.6; t_(R) (λ=220 nm):0.652 min (LC/MS—Method 2).

¹H-NMR (400 MHz, DMSO-d₆): δ=8.15 (t, 1H, J=5.6 Hz), 7.93 (t, 1H, J=5.6Hz), 5.96 (d, 1H, J=8.3 Hz), 5.43 (t, 1H, J=9.8 Hz), 5.08 (t, 1H, J=9.8hz), 4.98 (dd, 1H, J=9.8, 8.4 Hz), 4.30 (s, 1H, J=9.8 Hz), 3.49 (s, 4H),3.38 (q, 4H, J=6.0 Hz), 3.24-3.13 (m, 4H), 2.73 (t, 1H, J=2.6 Hz),2.37-2.31 (m, 2H), 2.30-2.24 (m, 2H), 2.07 (s, 3H), 2.00 (s, 3H), 1.95(s, 3H), 1.93 (s, 3H) ppm.

Example 106(3S,4S,5R)-3,4,5,6-Tetrahydroxy-N-[2-[2-[2-(pent-4-ynoylamino)ethoxy]ethoxy]ethyl]tetrahydropyran-2-carboxamide

Method G

To a solution of the tetraacetate derivative (1 eq.) inmethanol/water/THF (5/4/1, v/v/v) at 0° C. was added a solution of anaqueous 1 N lithium hydroxide solution (1 eq.). The reaction mixture wasagitated 15 minutes at 0° C., quenched with a 1 N aqueous solution ofHCl, evaporated, and lyophilised.

Example 106 was obtained from example 105 using acetyl deprotectionmethod G.

Yield: 62.2 mg (153 μmol, quant).

LC/MS (ES-API): m/z=405.2 [M+H]⁺; calculated: 404.4; t_(R) (λ=324 nm):0.087 min (LC/MS—Method 2).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.77 (d, 0.5H, J=6.7 Hz), 6.47 (d, 0.5H,J=4.6 Hz), 4.98-4.92 (m, 2H), 4.90 (d, 0.5H, J=4.3 Hz), 4.77 (d, 0.5H,J=4.9 Hz), 4.57 (d, 0.5H, J=6.3 Hz), 4.32 (t, 0.5H, J=7.4 Hz), 3.95 (t,0.5H, J=10 Hz), 3.50 (d, 4H, J=2.7 Hz), 3.49-3.37 (m, 4H), 3.26-3.09 (m,6H), 2.98-2.91 (m, 0.5H), 2.74 (t, 1H, J=2.6 Hz), 2.37-2.32 (m, 2H),2.99-2.45 (m, 2H) ppm. Mixture of diastereoisomers.

Example 107N-[[(3aR,5aS,8aS,8bR)-2,2,7,7-Tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[1,3]dioxolo[4,5-a:4′,5′-c]pyran-5-yl]methyl]oct-7-yn-1-amine

A solution of(3aR,5S,5aR,8aS,8bR)-2,2,7,7-tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[1,3]dioxolo[4,5-a:4′,5′-c]pyran-5-carbaldehyde(50 mg, 174 μmol, 1 eq.), oct-7-yn-1-amine hydrochloride (31 mg, 192μmol, 1.1 eq.), and triethylamine (27 μl, 192 μmol, 1.1 eq.) in methanol(1 mL) was agitated at room temperature for 3 hours. The reactionmixture was cooled to 0° C. and sodium boronhydride (13 mg, 348 μmol, 2eq.) was added. The reaction mixture was agitated at 0° C. for one hour.A 1 N aqueous solution of sodium hydroxide was added. The aqueous phasewas extracted with ether (three times), dried over MgSO₄, filtered, andevaporated in vacuo.

Purification: flash chromatography on silica gel (dichloromethane tilldichloromethane/methanol 9/1 in 30 min).

Yield: 29 mg (79 μmol, 45%), oil.

LC/MS (ES-API): m/z=368.4 [M+H]⁺; calculated: 368.5; t_(R): 0.684 min(LC/MS—Method 2).

¹H-NMR (400 MHz, DMSO-d₆): δ=5.43 (d, 1H, J=5.2 Hz), 4.55 (dd, 1H,J=7.7, 2.3 Hz), 4.44-4.39 (m, 0.3H), 4.30 (dd, 1H, J=5.1, 2.3 Hz), 4.20(dd, 1H, J=7, 7, 1.7 Hz), 3.74 (td, 1H, J=6.5, 1.5 Hz), 3.40-3.37 (m,0.7H), 2.72 (t, 0.6H, J=2.8 Hz), 2.68-2.65 (m, 0.3H), 2.63-2.58 (m,0.9H), 1.7 (t, 0.8H, J=2.5 Hz), 1.48-1.22 (m, 20.6H) ppm.

Example 108(3R,4S,5R)-6-[(Oct-7-ynylamino)methyl]tetrahydropyran-2,3,4,5-tetrol

Example 108 was obtained from example 107 following the isopropylidenedeprotection described in synthesis method F.

Yield: 32 mg (80 μmol, quant).

LC/MS (ES-API): m/z=288.2 [M+H]⁺; calculated: 288.2; t_(R)=0.648 min(LC/MS—Method 3).

¹H-NMR (400 MHz, DMSO-d₆): δ=6.71 (d, 0.5H, J=7.5 Hz), 6.38 (d, 0.5H,J=4.6 Hz), 5.00 (t, 0.5H, J=4.0 Hz), 4.91-4.79 (m, 2H), 4.71 (s, 0.5H),4.51 (s, 0.5H), 4.29 (t, 0.5H, J=6.8 Hz), 4.12-4.07 (m, 0.5H), 3.70-3.65(m, 1H), 3.63-3.51 (m, 1.5H), 3.19-3.00 (m, 2H), 2.95-2.83 (m, 2H), 2.75(t, 0.5H, J=2.6 Hz), 2.19-2.07 (m, 2H), 1.72 (t, 1H, J=2.6 Hz),1.64-1.52 (m, 2H), 1.48-1.52 (m, 6H) ppm. Mixture of diastereoisomers.

Example 109[(2S,3R,4S,5S)-2,3,5-Triacetoxy-6-(oct-7-ynylcarbamoyl)tetrahydropyran-4-yl]acetate

To a solution of (2R,3S,4S,5R)-2,3,4,5-tetraacetoxycyclohexanecarboxylicacid (20 mg, 55 μmol, 1 eq.) in dimethylformamide (2 mL) with 4 Åmolecular sieves was added HATU (29.4 mg, 77 μmol, 1.4 eq.) followed byoct-7-yn-1-amine hydrochloride (26.8 mg, 166 μmol, 3 eq.) in 0.5 mLdimethylformamide. The reaction mixture was agitated overnight at roomtemperature and evaporated in vacuo.

Purification: flash chromatography on silica gel, (heptane till ethylacetate in 20 min, 100% ethyl acetate for 20 min).

Yield: 25.5 mg (54 μmol, 98%), white solid.

LC/MS (ES-API): m/z=492.1 [M+H]⁺; calculated: 492.5; t_(R)=0.851 min(LC/MS—Method 2).

¹H-NMR (400 MHz, CDCl₃): δ=6.29 (t, 1H, J=4.9 Hz), 5.76 (d, 1H, J=8.0Hz), 5.30 (t, 1H, J=9.4 Hz), 5.19 (t, 1H J=9.4 Hz), 5.11 (td, 1H, J=8.0,1.5 Hz), 4.05 (d, 1H, J=9.4 Hz), 3.30-3.13 (m, 2H), 2.28 (td, 1H, J=7.0,2.6 Hz), 2.14 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.95(t, 1H, J=2.6 Hz), 1.78 (t, 1H, J=2.3 Hz), 1.58-1.22 (m, 8H) ppm.

Example 110(3S,4S,5R)-3,4,5,6-Tetrahydroxy-N-oct-7-ynyl-tetrahydropyran-2-carboxamide

Example 110 was obtained from example 109 using acetyl deprotectionmethod G.

Yield: 17 mg (49 μmol, 91%), orange oil.

LC/MS (ES-API): m/z=302.1 [M+H]⁺; calculated: 302.1; t_(R) (λ=220 nm):0.831 min (LC/MS—Method 3).

¹H-NMR (400 MHz, MeOD): δ=5.19 (s, 0.5H), 4.55 (d, 0.5H, J=7.5 Hz), 4.17(d, 0.7H, J=9.9 Hz), 3.71 (t, 1H, J=9.0H), 3.58 (s, 0.3H), 3.51-3.39 (m,1.7H), 3.27-3.18 (m, 3H), 2.18 (s, 3H), 2.12 (s, 1.1H), 1.92 (s, 0.9H),1.74 (s, 1.4H), 1.59-1.29 (m, 6H) ppm. Mixture of diastereoisomers.

Example 111 N-[6-[1-(4-(HumanInsulin-B29Lys-amino)-4-oxo-butyl)triazol-4-yl]hexyl]-(3S,4S,5R)-3,4,5,6-tetrahydroxy-tetrahydropyran-2-carboxamide

Step 1: 4-azido-butan-(human Insulin-B29Lys)-amide

To a solution of human insulin (300 mg, 51 μmol, 1 eq.) indimethylformamide/water (½, v/v, 9 mL) was added triethylamine (144 μl,1.03 mmol, 20 eq.) to get to pH=10.

The reaction mixture was cooled to 0° C. A solution of2,5-dioxopyrrolidin-1-yl 4-azidobutenoate (12.9 mg, 56 μmol, 1.1 eq.) indimethylformamide (1 mL) was added dropwise to the reaction mixture at0° C. over 10 minutes. The reaction mixture was agitated 2 hours,quenched with 1 N aqueous HCl till pH=3, and lyophilized.

Purification: AEKTA avant 25 (GE Healthcare), HPLC; column: KinetexPrep-C₁₈ column (5 μm, 250×21.1 mm, Phenomenex, volume 87 mL);wavelength for detection: 280 nm; eluent: (A) water+0.5% acetic acid,(B) 60/40 acetonitrile/water+0.5% acetic acid.

HPLC Gradient:

Flow rate Flow rate Column start % B end % B [cm/h] [mL/min] volume 0 080 4.7 2.0 0 100 105 6.2 14.0 100 100 105 6.2 2.0

Yield: 146.5 mg (24.75 μmol, 48%), white powder.

LC/MS (ES-API): m/z=1184.6 [M/5+H]⁺; calculated: 1184.7; t_(R) (λ=215nm): 4.30 min (LC/MS—Method 4).

Step 2: N-[6-[1-(4-(humanInsulin-B29Lys-amino)-4-oxo-butyl)triazol-4-yl]hexyl]-3,4,5,6-tetrahydroxy-tetrahydropyran-2-carboxamideMethod H

To a solution of the alkynes (1.2 eq.) and example 111, step 1 (1 eq.)in dimethylformamide and water was added a mixture of the click reagentspremixed in this order: CuSO₄*5H₂O (0.5 eq.), THPTA (0.8 eq.), andsodium ascorbate (1 eq.). The reaction mixture was agitated at roomtemperature for 2 hours. The reaction mixture was lyophilised.Purification was done on reverse phase chromatography.

Example 111, step 2 was obtained from example 111, step 1 and example110 following the click chemistry procedure described in synthesismethod H.

Purification and HPLC gradient like in example 111, step 1.

Yield: 2.9 mg (0.46 μmol, 18%), white powder.

LC/MS (ES-API): m/z=1244.9 [M/5+H]⁺; calculated: 1245.0; t_(R) (λ=215nm): 3.95 min (LC/MS—Method 4).

Example 1124-[4-[3-Oxo-3-[2-[2-[2-[[(3R,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methylamino]ethoxy]ethoxy]ethylamino]propyl]triazol-1-yl]butan-(humanInsulin-B29Lys)-amide

Example 112 was obtained from example 111, step 1 and example 114following the click chemistry procedure described in synthesis method H.

Purification and HPLC gradient like in example 111, step 1.

Yield: 1.37 mg (0.22 μmol, 11%), white powder.

LC/MS (ES-API): m/z=1262.7 [M/5+H]⁺; calculated: 1262.8; t_(R) (λ=215nm): 3.75 min (LC/MS—Method 4).

Example 1134-[4-[6-[[(3R,4S,5R)-3,4,5,6-Tetrahydroxytetrahydropyran-2-yl]methylamino]hexyl]triazol-1-yl]butan-(humanInsulin-B29Lys)-amide

Example 113 was obtained from example 111, step 1 and example 108following the click chemistry procedure described in synthesis method H.

Purification and HPLC gradient like in example 111, step 1.

Yield: 4.36 mg (0.70 μmol, 35%), white powder.

LC/MS (ES-API): m/z=1242.0 [M/5+H]⁺; calculated: 1242.2; t_(R) (λ=215nm): 3.77 min (LC/MS—Method 4).

Example 114 (3S,4S,5R)—N-[2-[2-[2-[3-[1-(4-(Humaninsulin-B29Lys)-4-oxo-butyl)triazol-4-yl]propanoylamino]ethoxy]ethoxy]ethyl]-3,4,5,6-tetrahydroxy-tetrahydropyran-2-carboxamide

Example 114 was obtained from example 111, step 1 and example 106following the click chemistry procedure described in synthesis method H.

Purification and HPLC gradient like in example 111, step 1.

Yield: 3.74 mg (0.59 μmol, 29%), white powder.

LC/MS (ES-API): m/z=1265.4[M/5+H]⁺; calculated: 1265.6; t_(R) (λ=215nm): 3.87 min (LC/MS—Method 4).

Example 1154-[4-[2-[2-[2-[4-[4-[[(3R,4S,5R)-3,4,5,6-Tetrahydroxytetrahydropyran-2-yl]methyl]piperazin-1-yl]phenoxy]ethoxy]ethoxy]ethoxymethyl]triazol-1-yl]butan-(humanInsulin-B29Lys)-amide

Example 115 was obtained from example 111, step 1 and example 100following the click chemistry procedure described in synthesis method H.

Purification and HPLC gradient like in example 111, step 1.

Yield: 15.2 mg (2.36 μmol, 28%), white powder.

LC/MS (ES-API): m/z=1072.3 [M/6+H]; calculated: 1072.5; t_(R) (λ=215nm): 3.81 min.

Example 116 Methy-6-O-p-toluylsulfonyl-β-D-glucopyranoside

To a solution of methyl-ß-D-glucopyranoside (15 g, 77.3 mmol) inpyridine (50 mL) a solution of toluenesulfonylchloride (19.1 g, 100.5mmol) in CH₂Cl₂ (50 mL) was added dropwise at 0° C. The mixture was thenleft for 16 h at 8° C. Methanol (200 mL) was added to the reaction andthe solvents evaporated under reduced pressure. The residue was purifiedby column chromatography on silica gel (10:1 CH₂Cl₂/MeOH) to give theproduct as a white solid

Yield: 13.1 g (49%).

LC-Mass Method: Mobile phase: A=2.5 mM TFA/H2O, B=2.5 mM TFA/MeCN;Gradient: B=10%-95% in 1 min; Flow rate: 1.5 mL/min; Column:Xbridge-C18, 30×4.6 mm, 2.5 um.). LC purity: 92% (214 nm);

Mass: find peak 370.8 (M+Na)+ at 1.60 min.

¹H NMR (400 MHz, DMSO-d6) δ 7.79 (d, J=8.0 Hz, 2H), 7.50 (d, J=8.0 Hz,2H), 5.23 (d, J=5.6 Hz 1H), 5.13 (d, J=5.2 Hz, 1H), 5.00 (d, J=5.2 Hz,1H), 4.20 (dd, J=10.4 Hz, 2.0 Hz, 1H), 4.06-4.02 (m, 2H), 3.36-3.34 (m,1H), 3.30 (s, 3H), 3.12-3.07 (m, 1H), 3.01-2.95 (m, 1H), 2.93-2.87 (m,1H), 2.43 (s, 3H).

Example 117 Methyl-6-O-(3-phenoxyphenylcarbonyl)-β-glucopyranoside

Sodium hydride (17.22 mg, 430.58 μmol) was added to a solution of3-phenoxybenzoic acid (92.24 mg, 430.58 μmol) in DMF (4 mL) at 0° C.under argon atmosphere. The reaction mixture was stirred for about 30min and methyl-6-O-p-toluenesulfonyl-β-D-glucopyranoside 116 (100 mg,287.05 μmol) was added and the reaction stirred for 16 h at 80° C. Thesolvent was evaporated under reduced pressure in vacuo and the residueextracted with CH₂Cl₂/H₂O (3×). The organic layer was dried, thesolvents evaporated and the product purified by HPLC.

Yield: 53 mg (47%)

LC/MS (ES-API): m/z=435.20 [M−H+formic acid]−; calculated: 435.16,

tR (λ=220 nm): 1.6 min (LC/MS—Method 2)

¹H NMR (400.23 MHz, DMSO-d6) δ ppm 7.72 (d, J=7.48 Hz, 1H), 7.56 (t,J=7.95, 7.95 Hz, 1H), 7.44 (m, 3H), 7.33 (ddd, J=8.19, 2.57, 0.86 Hz,1H), 7.21 (t, J=7.18, 7.18 Hz, 1H), 7.08 (d, J=7.84 Hz, 2H), 5.15 (br s,1H), 4.55 (dd, J=11.68, 2.02 Hz, 1H), 4.26 (dd, J=11.74, 6.48 Hz, 1H),4.08 (d, J=7.82 Hz, 1H), 3.46 (br s, 2H), 3.43 (u), 3.29 (s, 5H), 3.17(m, 3H), 2.97 (m, 1H).

Example 118 Methyl-6-O-(4-phenoxyphenylcarbonyl)-β-glucopyranoside

Methyl-6-O-(4-phenoxyphenylcarbonyl)-β-D-glucopyranoside was synthesizedas described for example 117 fromMethyl-6-O-p-toluylsulfonyl-β-D-glucopyranoside (116) (100 mg, 287.05μmol) and 4-phenoxybenzoic acid (92.24 mg, 430.58 μmol).

Yield: 49 mg (43.7%)

LC/MS (ES-API): m/z=435.22 [M−H+formic acid]⁻; calculated: 435.16,

tR (λ=220 nm): 1.6 min (LC/MS—Method 2)

¹H NMR (400.23 MHz, DMSO-d6) δ ppm 7.97 (m(para), 2H), 7.46 (t, J=7.56,7.56 Hz, 2H), 7.25 (m, 1H), 7.13 (d, J=7.83 Hz, 2H), 7.08 (m(para), 2H),4.54 (dd, J=11.80, 1.90 Hz, 1H), 4.29 (dd, J=11.80, 6.17 Hz, 1H), 4.11(d, J=7.82 Hz, 1H), 3.47 (m, 4H), 3.20 (m, 6H), 2.99 (m, 2H), 2.50 (u),2.33 (m, 1H)

Example 119Methyl-6-O-(2-Methyl-4-phenoxyphenylcarbonyl)-β-D-glucopyranoside

Methyl-6-O-(2-Methyl-4-phenoxyphenylcarbonyl)-β-D-glucopyranoside wassynthesized as described for example 117 fromMethyl-6-O-p-toluylsulfonyl-β-D-glucopyranoside (116; 100 mg, 287.05μmol) and 2-Methyl-4-phenoxybenzoic acid (98.28 mg, 430.58 μmol)

Yield: 46 mg (39.6%)

LC/MS (ES-API): m/z=499.17 [M−H+formic acid]⁻; calculated: 449.18,

tR (λ=220 nm): 1.63 min (LC/MS—Method 2)

Example 120Methyl-6-O-(3-chloro-3′-methoxy-4-phenoxyphenylcarbonyl)-β-D-glucopyranoside

Methyl-6-O-(3-chloro-3′-methoxy-4-phenoxyphenylcarbonyl)-β-D-glucopyranosidewas synthesized as described for example 117 fromMethyl-6-O-p-toluylsulfonyl-β-D-glucopyranoside (116; 100 mg, 287.05μmol) and 3-Chloro-3′-methoxy-4-phenoxybenzoic acid (120 mg, 430.58μmol).

Yield: 43 mg (32.9%)

LC/MS (ES-API): m/z=499.17 [M−H+formic acid]⁻; calculated: 499.13,

tR (λ=220 nm): 1.64 min (LC/MS—Method 2)

Example 121Methyl-6-O-(3-methoxy-4-phenoxyphenylcarbonyl)-β-D-glucopyranoside

Methyl-6-O-(3-methoxy-4-phenoxyphenylcarbonyl)-β-D-glucopyranoside wassynthesized as described for example 117 fromMethyl-6-O-p-toluylsulfonyl-β-D-glucopyranoside (116; 100 mg, 287.05μmol) and 3-Methoxy-4-phenoxybenzoic acid (105.17 mg, 430.58 mmol).

Yield: 58 mg (48.1%)

LC/MS (ES-API): m/z=465.14 [M−H+formic acid]⁻; calculated: 465.17,

tR (λ=220 nm): 1.53 min (LC/MS—Method 2)

¹H NMR (400.23 MHz, DMSO-d6) δ ppm 7.64 (d, J=1.96 Hz, 1H), 7.59 (dd,J=8.31, 1.96 Hz, 1H), 7.37 (t, J=7.64, 7.64 Hz, 2H), 7.13 (t, J=7.13,7.13 Hz, 1H), 7.04 (d, J=8.44 Hz, 1H), 6.96 (d, J=7.84 Hz, 2H), 5.24 (d,J=5.01 Hz, 1H), 5.11 (d, J=4.89 Hz, 1H), 5.05 (d, J=4.03 Hz, 1H), 4.59(dd, J=11.74, 1.96 Hz, 1H), 4.29 (dd, J=11.74, 6.48 Hz, 1H), 4.12 (d,J=7.70 Hz, 1H), 3.83 (s, 3H), 3.47 (u), 3.20 (m, 3H), 3.00 (m, 1H).

Example 122Methyl-6-O-(1-benzyl-3-bromo-2-oxo-1,2-dihydropyridinyl-4-carbonyl)-β-D-glucopyranoside

Methyl-6-O-(1-benzyl-3-bromo-2-oxo-1,2-dihydropyridinyl-4-carbonyl)-β-D-glucopyranosidewas synthesized as described for example 117 fromMethyl-6-O-p-toluylsulfonyl-β-D-glucopyranoside (116; 100 mg, 287.05μmol) and 1-benzyl-3-bromo-2-oxo-1,2-dihydropyridine-4-carboxylate (400mg, 649.07 μmol).

Yield: 8 mg

LC/MS (ES-API): m/z=484.09 [M+H]⁺; calculated: 484.06,

tR (λ=220 nm): 1.33 min (LC/MS—Method 2)

Example 123Methyl-2-O-(3-methoxy-4-phenoxyphenylcarbonyl)-α-D-glucopyranoside

Methyl-2-O-(3-methoxy-4-phenoxyphenylcarbonyl)-α-D-glucopyranoside wassynthesized according to a method described by Muramatsu and Takemotofrom Methyl-α-D-glucopyranoside and 3-Methoxy-4-phenoxybenzoic acid.3-Methoxy-4-phenoxybenzoic acid (182.27 mg, 669.47 μmol) is suspended inCH₂Cl₂ (3 mL) under Argon atmosphere.1-Chloro-N,N,2-trimethylpropenylamine (88.57 μl, 669.47 μmol) is addedand the reaction mixture stirred for 20 min at 20° C. to yield thecorresponding carboxylic acid chloride.

A solution of Methyl-α-D-glucopyranoside (97.05 mg, 257.49 μmol) andDibutyltinndichloride (16.47 mg, 51.50 μmol) in THF (3 mL) is stirredfor 15 min. Tetrabutylammoniumiodide (97.05 mg, 257.49 μmol) andDiisopropylethylamine (DIPEA; 113.85 μl, 669.47 μmol) are added.Subsequently the solution of 3-Methoxy-4-phenoxybenzoic acid chloride inCH₂Cl₂ is added, the reaction mixture stirred for 2 h and left for 18 hat 20° C.

The reaction is quenched with NH4Cl-solution and the product extractedwith EtOAc (3×5 mL). The product is finally purified by HPLC and freezedried.

Yield: 55 mg (25.4%)

LC/MS (ES-API): m/z=465.0 [M−H+formic acid]⁻ [M+H tR (λ=220 nm): 1.73min (LC/MS—Method 2)

¹H NMR (400.23 MHz, DMSO-d6) δ ppm 7.66 (m, 1H), 7.62 (d, J=1.83 Hz,1H), 7.37 (m, 2H), 7.13 (m, 1H), 7.06 (d, J=8.46 Hz, 1H), 6.95 (d,J=7.82 Hz, 2H), 4.88 (d, J=3.67 Hz, 1H), 4.64 (dd, J=9.96, 3.61 Hz, 1H),3.84 (s, 3H), 3.78 (m, 1H), 3.69 (br d, J=10.15 Hz, 1H), 3.52 (br dd,J=11.80, 5.56 Hz, 2H), 3.44 (u), 3.28 (s, 5H), 3.25 (m, 1H), 2.50 (u),2.33 (s, 1H)

Example 124Methyl-2-O-(3-chloro-3′-methoxy-4-phenoxyphenylcarbonyl)-α-D-glucopyranoside

Methyl-2-O-(3-chloro-3′-methoxy-4-phenoxyphenylcarbonyl)-α-D-glucopyranosidewas synthesized as described for example 123 fromMethyl-α-D-glucopyranoside (100 mg, 514.98 μmol) and3-Chloro-3′-methoxy-4-phenoxybenzoic acid (124.24 mg, 669.47 μmol).

Yield: 124 mg (50%)

LC/MS (ES-API): m/z=499.0 [M−H+formic acid]⁻ tR (λ=220 nm): 1.94 min(LC/MS—Method 2)

¹H NMR (400.23 MHz, DMSO-d6) δ ppm 7.80 (d, J=1.83 Hz, 1H), 7.67 (d,J=1.71 Hz, 1H), 7.31 (t, J=8.01, 8.01 Hz, 2H), 7.05 (t, J=7.40, 7.40 Hz,1H), 6.82 (d, J=7.95 Hz, 2H), 5.42 (br s, 1H), 5.22 (br s, 1H), 4.91 (d,J=3.55 Hz, 1H), 4.63 (m, 1H), 3.82 (s, 3H), 3.79 (m, 1H), 3.69 (br d,J=10.27 Hz, 1H), 3.53 (dd, J=11.80, 5.56 Hz, 1H), 3.44 (u), 3.29 (s,5H), 3.27 (m, 1H), 3.20 (br s, 1H), 2.33 (s, 1H)

Synthesis of Allyl-6-O-toluenesulfonyl-β-D-glucopyranoside

Allyl-6-O-toluenesulfonyl-β-D-glucopyranoside was synthesized accordingto a published procedure [R. Brisco et al., Carb. Res. 348 (2012),27-32] starting from commercially available allyl-β-D-glucopyranoside.

To a solution of allyl-β-D-glucopyranoside (1 g, 4.54 mmol) in pyridine(30 mL) was added p-toluenesulfonylchloride ((1.47 g, 7.72 mmol) at 0°C. The reaction mixture was stirred for 30 min and then stored at 0° C.for 16 h. The reaction was controlled by TLC (9:1, CH₂Cl₂/MeOH) provingconsumption of starting material. The reaction mixture was quenched withMeOH and the solvents were removed under reduced pressure. The Productwas purified by flash chromatography (EtOAc/MeOH, 9:1).

Yield: 850 mg (51%)

Example 125 Allyl-6-O-(3-phenoxyphenylcarbonyl)-β-glucopyranoside

Allyl-6-O-(3-phenoxyphenylcarbonyl)-β-D-glucopyranoside was synthesizedfrom allyl-6-O-toluenesulfonyl-β-D-glucopyranoside (200 mg, 534.18 μmol)and 3-phenoxyphenylcarboxylic acid (171.65 mg, 801.27 μmol) as describedfor example 117.

Yield: 113 mg (50.8%)

LC/MS (ES-API): m/z=461.25 [M−H+formic acid]⁻; calculated: 461.18 tR(λ=220 nm): 1.69 min (LC/MS—Method 2)

¹H NMR (400.23 MHz, DMSO-d6) δ ppm 7.73 (d, J=7.44 Hz, 1H), 7.57 (t,J=7.95, 7.95 Hz, 1H), 7.48 (s, 1H), 7.43 (t, J=7.27, 7.27 Hz, 2H), 7.33(ddd, J=8.19, 2.57, 0.98 Hz, 1H), 7.20 (m, 1H), 7.07 (d, J=7.78 Hz, 2H),5.85 (m, 1H), 5.23 (m, 2H), 5.09 (m, 3H), 4.54 (dd, J=11.74, 1.96 Hz,1H), 4.28 (dd, J=11.74, 6.60 Hz, 1H), 4.16 (m, 2H), 3.98 (m, 1H), 3.44(u), 3.17 (m, 2H), 3.01 (td, J=8.34, 8.34, 4.95 Hz, 1H).

Example 126 Allyl-6-O-(4-phenoxyphenylcarbonyl)-β-glucopyranoside

Allyl-6-O-(4-phenoxyphenylcarbonyl)-3-D-glucopyranoside was synthesizedfrom allyl-6-O-toluenesulfonyl-β-D-glucopyranoside (200 mg, 534.18 μmol)and 3-phenoxyphenylcarboxylic acid (171.65 mg, 801.27 μmol) as describedfor example 117.

Yield: 116 mg (52.1%)

LC/MS (ES-API): m/z=461.20 [M−H+formic acid]⁻; calculated: 461.18 tR(λ=220 nm): 1.70 min (LC/MS—Method 2)

¹H NMR (400.23 MHz, DMSO-d6) δ ppm 7.98 (m(para), 2H), 7.46 (t, J=7.26,7.26 Hz, 2H), 7.25 (t, J=7.47, 7.47 Hz, 1H), 7.13 (d, J=7.84 Hz, 2H),7.08 (m(para), 2H), 5.87 (m, 1H), 5.26 (m, 2H), 5.09 (br dd, J=10.39,1.83 Hz, 2H), 4.53 (dd, J=11.74, 1.96 Hz, 1H), 4.22 (m, 3H), 4.02 (m,1H), 3.46 (u), 3.20 (m, 4H), 3.03 (m, 1H).

Synthesis of Trimethylsilylethoxy-6-O-tosyl-β-D-glucopyranoside

LCMS Conditions:

LCMS-Condition 01: Method:—LCMS_X-Select (Formic Acid)

Column: X-Select CSH C18 (4.6*50) mm 2.5 u, Mobile Phase: A.0.1% Formicacid in water B. 0.1% Formic acid in Acetonitrile Inj Volume; 5.0 μL,Flow Rate: 1.0 mL/minute, Gradient program: 2% B to 98% B in 2.8 minute,Hold till 4.8 min, At 5.0 min B conc is 2% up to 7.0 min.

ELSD Conditions:

ELSD-Condition 01: Method:—LCMS_X-Bridge (NH₃)

Column: X-Bridge C18 (4.6*50) mm 3.5μ; Mobile Phase: A. 0.05% NH3 inwater. B: 0.05% NH3 in Acetonitrile Inj Volume; 0.2 μL, Flow Rate: 1.200mL/minute; Gradient program: 2% B to 100% B in 3.5 minute, Hold till 4.5min, At 4.7 min B conc is 2% up to 6.0 min.

Step-1: Synthesis of(2R,3R,4S,5R,6R)-6-(acetoxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate (2)

To (2S,3R,4S,5S,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol1 (50 g, 277.7 mmol) in pyridine (500 mL) at 0° C. was added DMAP (339mg, 2.777 mmol) and acetic anhydride (500 mL). The reaction mixture wasfurther stirred at room temperature for 20 h. After completion of thereaction, the pyridine was evaporated under reduced pressure and theresidue was diluted with water and extracted with CH₂Cl₂ (thrice). Thecombined organic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. The crude compound was purified bytrituration with n-hexane to afford 100 g (92% yield) of compound 2 asoff white solid.

ELSD-Condition-1: [M+H]⁺=408.00; Rt=2.88 min

¹H NMR (400 MHz, CDCl₃) δ: 6.33 (d, J=3.91 Hz, 1H), 5.47 (t, J=10.03 Hz,1H), 5.07-5.17 (m, 2H), 4.24-4.29 (m, 1H), 4.08-4.15 (m, 2H), 2.18 (s,3H), 2.09 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H).

Step-2: Synthesis of(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyltriacetate (3)

To (2R,3R,4S,5R,6R)-6-(acetoxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate 1 (25 g, 64.04 mmol) in CH₂Cl₂ (500 mL) at 0° C. was added33% solution of HBr in acetic acid (250 mL) and stirred for 4 h. Aftercompletion of the reaction, the reaction mixture was poured over ice andthe organic layer was separated and washed with water (400 mL) followedby saturated NaHCO₃ (200 mL×2). The combined organic layer was driedover anhydrous Na₂SO₄, filtered and concentrated under reduced pressure.The crude compound was purified by silica gel column chromatographyeluting with 0-50% ethyl acetate in n-hexane to afford 24 g (89% yield)of compound 3 as off white solid.

¹H NMR (400 MHz, CDCl₃): 6.61 (d, J=3.91 Hz, 1H), 5.56 (t, J=9.54 Hz,1H), 5.17 (t, J=9.78 Hz, 1H), 4.84 (dd, J=3.91, 10.27 Hz, 1H), 4.28-4.36(m, 2H), 4.10-4.16 (m, 1H), 2.10 (s, 3H), 2.10 (s, 3H), 2.06 (s, 3H),2.04 (s, 3H).

Step-3: Synthesis of(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(trimethylsilyl)ethoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (4)

To a stirred solution of HgO (12.1 g, 55.93 mmol), HgBr₂ (700 mgcatalytic), CaSO₄ (15.2 g, 111.86 mmol) and 2-(trimethylsilyl)ethan-1-ol(9.9 g, 83.90 mmol) in CHCl₃ (184 mL) was added(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyltriacetate 3 (23 g, 55.93 mmol) and stirred at room temperature for 1 h.After completion of the reaction, the reaction mixture was filteredthrough a pad of Celite and washed with CH₂Cl₂ and washed with saturatedNaHCO₃ solution. The combined organic layer was dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure. The crudecompound was purified by silica gel column chromatography eluting with0-30% ethyl acetate in n-hexane to afford 20 g (83% yield) of compound 4as colorless oil.

¹H NMR (400 MHz, CDCl₃): 5.19 (dd, J=8.07, 9.54 Hz, 1H), 5.08 (dd,J=8.07, 9.54 Hz, 1H), 4.97 (dd, J=8.07, 9.54 Hz, 1H), 4.51 (d, J=8.31Hz, 1H), 4.23-4.29 (m, 1H), 4.09-4.16 (m, 2H), 3.97 (dt, J=5.87, 10.03Hz, 1H), 3.66-3.76 (m, 2H), 3.52-3.60 (m, 1H), 2.08 (s, 2H), 2.03-2.04(m, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.25 (t, J=7.09 Hz, 1H), 0.85-0.98(m, 3H), 0.02 (s, 3H), 0.00 (s, 6H).

Step-4: Synthesis of(2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-(2-(trimethylsilyl)ethoxy)tetrahydro-2H-pyran-3,4,5-triol(5)

To(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(trimethylsilyl)ethoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate 4 (10 g, 22.32 mmol) in methanol (100 mL) was added solutionof sodium methoxide (100 mg sodium in 7.5 mL methanol) and stirred atroom temperature for 12 h. After completion of the reaction, thepyridine was evaporated under reduced pressure and the residue wasstirred in diethyl ether. The solid precipitated out was filtered anddried to afford 4.2 g (67% yield) of 5 as off white solid.

¹H NMR (400 MHz, D₂O) δ: 4.34 (d, J=7.83 Hz, 1H), 3.87-3.96 (m, 1H),3.78 (d, J=11.74 Hz, 1H), 3.55-3.68 (m, 2H), 3.22-3.38 (m, 4H), 3.11 (t,J=8.56 Hz, 1H), 0.80-1.00 (m, 2H), −0.10 (s, 9H).

Step-5: Synthesis of((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(2-(trimethylsilyl)ethoxy)tetrahydro-2H-pyran-2-yl)methyl4-methylbenzenesulfonate (2016-00144)

To(2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-(2-(trimethylsilyl)ethoxy)tetrahydro-2H-pyran-3,4,5-triol5 (9.5 g, 33.92 mmol) in pyridine (95 mL) was added DMAP (413 mg, 3.392mmol) and tosyl chloride (7.1 g, 37.32 mmol) stirred at room temperaturefor 12 h. After completion of the reaction, the pyridine was evaporatedunder reduced pressure and the residue was diluted with water andextracted with ethyl acetate. The combined organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Thecrude compound was purified by silica gel column chromatography elutingwith 0-10% methanol in CH₂Cl₂ to afford 10.2 g (69% yield) of 2016-00144as off white solid.

LCMS-Condition-1: [M+18]+=452.15; Rt=1.78 min

¹H NMR (400 MHz, DMSO-d₆) δ: 7.76 (d, J=8.31 Hz, 2H), 7.47 (d, J=8.31Hz, 2H), 5.16 (d, J=5.38 Hz, 1H), 4.99 (dd, J=4.89, 9.29 Hz, 2H), 4.21(d, J=10.27 Hz, 1H), 4.12 (d, J=7.83 Hz, 1H), 4.02 (dd, J=6.60, 10.03Hz, 1H), 3.69-3.79 (m, 1H), 3.43-3.52 (m, 1H), 3.35 (br. s, 1H),3.05-3.13 (m, 1H), 2.95-3.03 (m, 1H), 2.84-2.92 (m, 1H), 2.42 (s, 3H),0.82-0.95 (m, 2H), 0.00 (s, 9H).

Example 127trimethylsilylethyl-6-O-(2-methyl-3-phenoxyphenylcarbonyl)-β-D-glucopyranoside

Trimethylsilylethyl-6-O-(2-methyl-3-phenoxyphenylcarbonyl)-β-D-glucopyranosidewas synthesized as described for example 117 fromtrimethylsilylethoxy-6-O-tosyl-β-D-glucopyranoside (100 mg, 230.11 μmol)and 2-methyl-3-phenoxybenzoic acid (78.78 mg (345.16 μmol).

Yield: 63 mg (55.8%)

LC/MS (ES-API): m/z=535.23 (M−H+formic acid]⁻; calculated: 535.23;

tR (λ=220 nm): 1.94 min (LC/MS—Method 2)

¹H NMR (400.23 MHz, DMSO-d6) δ ppm 7.95 (d, J=8.68 Hz, 1H), 7.52 (t,J=7.23, 7.23 Hz, 2H), 7.31 (t, J=7.14, 7.14 Hz, 1H), 7.17 (d, J=7.82 Hz,2H), 7.00 (d, J=2.32 Hz, 1H), 6.89 (dd, J=8.68, 2.45 Hz, 1H), 5.28 (brs, 1H), 5.07 (br s, 2H), 4.60 (dd, J=11.68, 2.02 Hz, 1H), 4.33 (dd,J=11.68, 7.15 Hz, 1H), 4.27 (d, J=7.82 Hz, 1H), 3.84 (ddd, J=11.06,9.78, 6.05 Hz, 1H), 3.55 (m, 2H), 3.24 (quin, J=8.71, 8.71, 8.71, 8.71Hz, 2H), 3.05 (t, J=8.19, 8.19 Hz, 1H), 2.58 (m, 10H), 2.50 (u), 0.95(m, 2H).

Example 128Trimethylsilylethyl-6-O-(3-chloro-3′-methoxy-4-phenoxyphenylcarbonyl)-β-D-glucopyranoside

trimethylsilylethyl-6-O-(3-chloro-3′-methoxy-4-phenoxyphenylcarbonyl)-β-D-glucopyranosidewas synthesized as described for example 117 fromtrimethylsilylethoxy-6-O-tosyl-β-D-glucopyranoside (100 mg, 230.11 μmol)and 2-methyl-3-phenoxybenzoic acid (78.78 mg, 345.16 μmol).

Yield: 64 mg (51.4%)

LC/MS (ES-API): m/z=585.15 [M−H+formic acid]⁻; calculated: 585.19 tR(λ=220 nm): 1.92 min (LC/MS—Method 2)

¹H NMR (400.23 MHz, DMSO-d6) δ ppm 7.79 (d, J=1.83 Hz, 1H), 7.72 (d,J=1.83 Hz, 1H), 7.39 (t, J=7.24, 7.24 Hz, 2H), 7.13 (t, J=7.34, 7.34 Hz,1H), 6.88 (d, J=7.82 Hz, 2H), 5.34 (br s, 1H), 5.10 (br s, 1H), 4.66(dd, J=11.62, 1.96 Hz, 1H), 4.45 (dd, J=11.68, 7.15 Hz, 1H), 4.30 (d,J=7.82 Hz, 1H), 3.89 (s, 3H), 3.84 (m, 1H), 3.60 (m, 2H), 3.56 (s, 1H),3.26 (m, 2H), 3.07 (br t, J=8.13, 8.13 Hz, 1H), 2.58 (dt, J=3.61, 1.74,1.74 Hz, 16H), 2.50 (u), 0.95 (m, 2H), 0.08 (s, 1H)

Example 129Trimethylsilylethyl-6-O-(3-methoxy-4-phenoxyphenylcarbonyl)-β-D-glucopyranoside

Trimethylsilylethyl-6-O-(3-methoxy-4-phenoxyphenylcarbonyl)-3-D-glucopyranosidewas synthesized as described for example 117 fromtrimethylsilylethoxy-6-O-toluenesulfonyl-β-D-glucopyranoside (100 mg,230.11 μmol) and 2-methyl-3-phenoxybenzoic acid (84.31 mg, 345.16 μmol).

Yield: 73 mg (52.6%)

LC/MS (ES-API): m/z=551.16 [M−H+formic acid]⁻; calculated: 551.23 tR(λ=220 nm): 1.84 min (LC/MS—Method 2)

Example 130 6-O-(3-phenoxyphenylcarbonyl)-D-glucopyranose

To a solution of allyl-6-O-(3-phenoxyphenylcarbonyl)-β-D-glucopyranoside(Example 125; 110 mg, 264.15 μmol) in MeOH (2 mL) Pd (II)chloride (9.37mg, 52.83 μmol) was added. After 3 h at 25° C. the reaction wascontrolled by LC/MS. A product with the desired mass could be detected.MeOH was added (4 mL) and the product was purified by HPLC.

Yield: 51 mg (53%, mixture of anomers)

LC/MS (ES-API): m/z=375.14 [M−H+formic acid]⁻; calculated: 375.12 tR(λ=220 nm): 1.45/1.43 min (Mixture of anomers, LC/MS—Method 2)

¹H NMR (400.23 MHz, DMSO-d6) δ ppm 7.72 (m, 1H), 7.48 (m, 4H), 7.31(ddd, J=8.10, 2.54, 0.86 Hz, 1H), 7.21 (m, 1H), 7.08 (m, 2H), 4.91 (d,J=3.55 Hz, 1H), 4.52 (dd, J=11.74, 1.83 Hz, 1H), 4.47 (dd, J=11.68, 1.90Hz, 1H), 4.31 (m, 2H), 3.88 (m, 1H), 3.48 (br s, 4H), 3.16 (m, 4H).

Example 131 6-O-(4-Phenoxyphenylcarbonyl)-D-glucopyranose

6-O-(4-phenoxyphenylcarbonyl)-D-glucopyranose was synthesized asdescribed for example 129 fromAllyl-6-O-(4-phenoxyphenylcarbonyl)-β-D-glucopyranoside (Example 126;110 mg, 264.15 mg) as described for Example 129.

Yield: 46 mg (46.3%, mixture of anomers)

LC/MS (ES-API): m/z=375.09 [M−H]⁻; calculated: 375.12 tR (λ=220 nm):1.46/1.44 min (Mixture of anomers, LC/MS—Method 2)

Example 132 6-O-(2-Methyl-4-phenoxyphenylcarbonyl)-D-glucopyranose

To a solution ofTrimethylsilylethyl-6-O-(2-methyl-3-phenoxyphenylcarbonyl)-β-D-glucopyranoside(Example 127, 60 mg, 122.29 μmol) in CH₂Cl₂ (1.8 mL) TFA (200 μl, 2.60mmol) was added under Argon atmosphere wurde. After 5 h LC/MS analysisshowed consumption of starting material and one new peak could bedetected. The reaction mixture was diluted with Water and freeze dried.The product was purified by HPLC.

Yield: 42 mg (88% mixture of anomers)

LC/MS (ES-API): m/z=389.16 [M−H]⁻; calculated: 389.13 tR (λ=220 nm):1.53/1.51 min (Mixture of anomers, LC/MS—Method 2)

Example 1336-O-(3-Chloro-3′methyloxy-4-phenoxyphenylcarbonyl)-D-glucopyranose

6-O-(3-Chloro-3′methyloxy-4-phenoxyphenylcarbonyl)-D-glucopyranose wassynthesized fromtrimethylsilylethyl-6-O-(3-chloro-3′-methoxy-4-phenoxyphenylcarbonyl)-β-D-glucopyranoside(128; 60 mg, 110.89 μmol) as described for example 131

Yield: 42 mg (85.9%, mixture of anomers)

LC/MS (ES-API): m/z=485.1/487.0 [M−H+formic acid]⁻; calculated: 485.12tR (λ=220 nm): 1.55/1.53 min (Mixture of anomers, LC/MS—Method 2)

Example 134 6-O-(4-Hydroxy-2-methyl-phenylcarbonyl)-D-glucopyranose

6-O-(3-Chloro-3′methyloxy-4-phenoxyphenylcarbonyl)-D-glucopyranose wassynthesized fromtrimethylsilylethyl-6-O-(4-hydroxy-2-methylphenyl)carbonyl-β-D-glucopyranoside(21 mg, 50.66 μmol) as described for example 131

Yield: 16 mg (quantitative, mixture of anomers)

LC/MS (ES-API): m/z=313.06 [M−H]⁻; calculated: 313.10 tR (λ=220 nm):0.72/0.64 min (Mixture of anomers, LC/MS—Method 2)

¹H NMR (400.23 MHz, DMSO-d6) δ ppm 10.11 (d, J=4.77 Hz, 1H), 7.75 (d,J=7.58 Hz, 1H), 6.67 (m, 2H), 6.32 (brd, J=3.55 Hz, 1H), 5.14 (brs, 1H),5.08 (brs, 1H), 4.92 (br s, 1H), 4.74 (br s, 1H), 4.50 (br s, 1H), 4.43(ddd, J=17.76, 11.71, 1.71 Hz, 1H), 4.33 (br s, 1H), 4.20 (m, 1H), 3.87(m, 1H), 3.48 (u), 3.16 (m, 3H), 2.92 (br t, J=8.19, 8.19 Hz, 1H).

Example 135 6-O-(3-Methyloxy-4-phenoxyphenylcarbonyl)-D-glucopyranose

6-O-(3-Methyloxy-4-phenoxyphenylcarbonyl)-D-glucopyranose wassynthesized fromtrimethylsilylethyl-6-O-(3-chloro-3′-methoxy-4-phenoxyphenylcarbonyl)-β-D-glucopyranoside(128; 60 mg, 110.89 μmol) as described for example 131

Yield: 42 mg (85.9%, mixture of anomers)

LC/MS (ES-API): m/z=405.09 [M−H]⁻; calculated: 405.13 tR (λ=220 nm):1.43/1.41 min (Mixture of anomers, LC/MS—Method 2)

¹H NMR (400.23 MHz, DMSO-d6) δ ppm 7.64 (m, 1H), 7.58 (d, J=8.60 Hz,1H), 7.37 (t, J=7.64, 7.64 Hz, 2H), 7.13 (t, J=7.04, 7.04 Hz, 1H), 7.06(d, J=8.58 Hz, 1H), 6.95 (d, J=8.44 Hz, 2H), 4.93 (d, J=3.42 Hz, 1H),4.51 (m, 1H), 4.32 (m, 1H), 3.91 (br dd, J=9.90, 3.91 Hz, 1H), 3.84 (s,3H), 3.49 (brs, 2H), 3.46 (brd, J=9.17 Hz, 3H), 3.18 (m, 5H), 2.93 (brt, J=8.19, 8.19 Hz, 1H), 2.50 (u), 2.33 (br s, 1H)

Example 1361,2:3,4-Diisopropyliden-6-O-(3-Methoxy-4-phenoxy)-phenylcarbonyl-D-glucopyranose

Lit: Angew. Chem. Int. Ed. 2008, 47, 8264-8267

A solution of 3-Methoxy-4-phenoxybenzoic acid (93.84 mg, 384.19 μmol) inCH2Cl2 (1.5 ml) was stirred under argon atmosphere at 0° C.Consecutively a solution of 1,2:3,4-Di-O-isopropyliden-D-galactose (100mg, 384.19 μmol) in DMF (1 mL), DMAP (9.39 mg, 76.84 μmol) and finallyDCC (79.27 mg, 384.19 μmol) where added and the reaction was kept at 0°C. for 10 min. The reaction mixture was left at 25° C. for 16 h. Thedesired mass could be detected by LC/MS. The reaction mixture wasdiluted with MeOH (2 mL) filtrated and the product finally purified byHPLC,

Yield: 154 mg (82.4%)

LC/MS (ES-API): m/z=487.20 [M−H]⁻; calculated: 487.19 tR (λ=220 nm):2.72 min (LC/MS—Method 2)

¹H NMR (400.23 MHz, DMSO-d6) δ ppm 7.63 (d, J=1.71 Hz, 1H), 7.57 (dd,J=8.38, 0.90 Hz, 1H), 7.38 (t, J=7.95, 7.95 Hz, 2H), 7.14 (t, J=7.16,7.16 Hz, 1H), 7.00 (m, 3H), 0.48 (d, J=5.01 Hz, 1H), 4.65 (dd, J=7.95,2.32 Hz, 1H), 4.36 (m, 4H), 4.12 (m, 1H), 3.83 (s, 3H), 3.46 (u), 2.50(u), 1.44 (s, 3H), 1.39 (s, 3H), 1.31 (s, 3H), 1.28 (s, 3H)

Example 137 6-O-(3-Methoxy-4-phenoxyphenylcarbonyl)-D-glucopyranose

1,2:3,4-Diisopropyliden-6-O-(3-methoxy-4-phenoxyphenylcarbonyl)-D-glucopyranose(154 mg, 316.54 μmol) was dissolved in acetonitril. Under argonatmosphere HCl (2.37 ml, 4.75 mmol) was added and the Reaction mixturewas stirred at 40° C. for 16 h. Water was added and the product wasfreeze dried. The product was finally purified by HPLC.

Yield: 51 mg (39.6%) (Mixture of Anomers)

LC/MS (ES-API): m/z=389.1 [M+H—H₂O]+; calculated: 389.11 tR (λ=220 nm):1.58/1.60 min (LC/MS—Method 2)

Example 1386-O-(3-Chloro-3′Methoxy-4-phenoxyphenylcarbonyl)-D-glucopyranose

6-O-(3-Chloro-3′Methoxy-4-phenoxyphenylcarbonyl)-D-glucopyranose wassynthesized as described for example 136 from1,2:3,4-Diisopropyliden-6-O-(3-chloro-3′methoxy-4-phenoxy)phenylcarbonyl-D-glucopyranose(150 mg, 287.93 μmol).

Yield: 93 mg (73.3%).

LC/MS (ES-API): m/z=423.1/425.1 [M+H—H₂O]⁺; calculated: 423.07 tR (λ=220nm): 1.76/1.78 min (LC/MS—Method 2)

Example 139 Step 1: Amide Coupling4-(2,4-Dichlorophenyl)piperazin-1yl-(1,2:3,4-diisopropyliden)-D-galacturonicacid amide

A solution of 1,2:3,4-Diisopropyliden-D-galacturonic acid (150 mg,546.91 μmol) in DMF (5 ml) under argon atmosphere was treated with HATU(291.14 mg, 765.67 μmol) and the reaction mixture stirred for 5 min.1-(2,4-dichlorophenyl)piperazine (176.96 mg, 765.67 μmol) was added andthe reaction mixture was stirred at 25° C. The reaction was monitored byLCMS. After 24 h starting material could be detected, DIPEA (2.2equivalents) was added and the reaction mixture left for another 24 h at25° C. CH2Cl2 (1×10 ml) and water were added and the product extracted.The organic layer was dried, the solvents evaporated and the productpurified by HPLC.

Yield: 163 mg (61.2%)

Example 140 Step 2: Deprotection4-(2,4-Dichlorophenyl)piperazin-1-yl-D-galacturonic acid amide

A suspension of4-(2,4-Dichlorophenyl)piperazin-lyl-(1,2:3,4-Diisopropyliden)-D-galacturonicacid amide (163 mg, 334.45 μmol) was treated with aqueous HCl (2.51 ml,5.02 mmol) under argon atmosphere and left for 16 h at 25° C. Thereaction was monitored by LC/MS, starting material could still bedetected. The reaction mixture was dissolved in MeOH and again treatedwith HCl (2.5 mL) and left at 25° C. for 16 h. Methanol was evaporated,the residue diluted with water and freeze dried. The product waspurified by HPLC.

Yield: 46 mg (28.3%)

LC/MS (ES-API): m/z=407.1/409.0/411. [M+H]+; calculated: 407.24 tR(λ=220 nm): 1.46/1.48 min (LC/MS—Method 2)

Example 140 Step 1: Preparation of substituted amine(R)-2-((benzyloxy)methyl)-4-chloro-piperazine

Lit.: ACS Med. Chem. Lett., 2015, 6 (10), pp 1041-1046

A solution of tert-butyl(R)-2-((benzyloxy)methyl)piperazine-1-carboxylate (333.98 mg, 1.09mmol), 1-bromo-4-chlorobenzene (227.46 mg, 1.19 mmol) and BINAP (40.72mg, 65.40 μmol) in toluene (5 ml) was treated with Pd(II)acetate (9.79mg, 43.60 μmol) under argon atmosphere. KoTBu (183.47 mg, 1.64 mmol) wasadded and the reaction mixture was irradiated in the microwave for 30min at 130° C.

EtOAc and H₂O where added, the reaction mixture filtered, and thefiltrate washed with brine. The solvent was evaporated and the residuepurified by HPLC.

Yield: 142 mg (41%)

Step 2: Reductive Amination6-Desoxy-6-((R)-2-((benzyloxy)methyl)-4-chlorophenyl)-piperazin-1-yl-(1,2:3,4-diisopropyliden)-D-galactopyranose

To a solution of(3aR,5S,5aR,8aS,8bR)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4′,5′-d]pyran-5-carbaldehyde(125.54 mg, 486.07 μmol) in methanol (2 ml),(R)-3-((benzyloxy)methyl)-1-(4-chlorophenyl)piperazine (140 mg, 441.88μmol), acetic acid (50.59 μl, 883.76 μmol) and sodiumcyanoborhydride(58.46 mg, 883.76 μmol) are added. The reaction mixture was left for 16h at 25° C. LCMS showed consumption of starting material. The reactionmixture was evaporated and the product purified by HPLC.

Yield: 105 mg (40%)

Step 3: DeprotectionMethyl-6-desoxy-6-((R)-2-((benzyloxy)methyl)-4-chlorophenyl)-piperazin-1-yI-D-galactopyranoside

A suspension of6-desoxy-6-(4-chlorophenyl)piperazino-(1,2:3,4-diisopropyliden)-D-galactopyranose(103 mg, 172.95 μmol) in MeOH (2 mL) is treated with mit HCl (1.73 ml,3.46 mmol) and stirred for 2 h and left for 16 h at 25° C.

Yield: 13 mg (12.4%)

LC/MS (ES-API): m/z=479.3/481.3. [M+H]+; calculated: 478.20 tR (λ=220nm): 1.37 (LC/MS—Method 2)

1H NMR (400.23 MHz, DMSO-d6) δ ppm 9.62 (br s, 1H), 7.35 (m, 8H), 7.02(br d, J=8.80 Hz, 2H), 5.05 (br s, 1H), 4.93 (br s, 1H), 4.58 (m, 3H),4.38 (br d, J=5.50 Hz, 1H), 4.28 (br d, J=10.03 Hz, 1H), 3.95 (br s,1H), 3.89 (br d, J=10.64 Hz, 2H), 3.77 (m, 4H), 3.64 (brd, J=6.85 Hz,1H), 3.57 (m, 4H), 3.17 (brs, 1H), 3.01 (m, 2H), 2.50 (u) 1H NMR (250.13MHz, DMSO-d6) δ ppm 7.29 (m, 6H), 6.92 (m, 2H), 5.02 (m, 1H), 4.55 (s,2H), 3.80 (m, 4H), 3.63 (m, 1H), 3.52 (brs, 2H), 3.18 (m, 7H), 2.93(brs, 97H), 2.75 (br s, 4H), 2.50 (u).

Synthesis of Example 141 Step 1: Reductive Amination6-Desoxy-6-(3-hydroxyphenyl)-piperazin-1-yl-(1,2:3,4-diisopropyliden)-D-galactopyranose

(3aR,5S,5aR,8aS,8bR)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4′,5′-d]pyran-5-carbaldehyde(239.09 mg, 925.74 μmol) was dissolved in MeOH (3 ml).3-(piperazin-1-yl)phenol (150 mg, 841.59 μmol), acetic acid, (96.36 μl,1.68 mmol) and NaCNBH3 (111.34 mg, 1.68 mmol) where added and thereaction mixture stirred for 3 h and left for 16 h at 25° C. Thereaction was monitored by LCMS. Starting material was consumed. Thesolvent was evaporated, the residue taken up in EtOAc/H2O and theproduct extracted with EtOAc and finally purified by HPLC.

Yield: 280 mg (72.8%)

Step 2: Activation of Spacer Undec-10-yn-1-yl 4-methylbenzenesulfonate

Lit.: Tetrahedron 56 (2000) p 1233-1245

Undec-10-yn-1-ol (343.17 μl, 2.97 mmol) was dissolved in CH₂Cl₂ (10 ml)under argon atmosphere. 4-Methylbenzenesulfonyl chloride (810.01 mg,4.25 mmol) was added and the reaction mixture cooled to 0° C. Pyridine(355.67 μl, 4.40 mmol) was added dropwise over 5 min and the reactionmixture was stirred for 3 h at 0° C. The ice bath was removed and thesolution was left for 16 h at 25° C. The reaction was monitored by LCMS.1 N HCl was added and the product extracted with CH₂Cl₂. The solvent wasremoved in vacuo and the product purified by HPLC

Yield: 1.57 g (29.3%)

Step 3: Ether Coupling6-Desoxy-6-(3-undecenyloxyphenyl)-4-piperazin-1-yl-(1,2:3,4-diisopropyliden)-D-galactopyranose

A mixture of6-desoxy-6-(3-hydroxyphenyl)-piperazin-1-yl-(1,2:3,4-diisopropyliden)-D-galactopyranoseHydrochlorid (275 mg, 601.80 μmol) and undec-10-yn-1-yl4-methylbenzenesulfonate (445.78 mg, 842.52 μmol) was treated withCesiumcarbonat (980.39 mg, 3.01 mmol). DMF (2 mL) was added and thereaction mixture was irradiated in the microwave at 80° C. for 1 h. LCMSshowed the expected mass. The product was extracted with EtOAc/H₂O, theorganic layer washed with aqueous NaCL solution (10%) and dried. Thesolvent was evaporated in vacuo and the product purified by HPLC.

Yield: 105 mg (28.7%)

Step 3: Deprotection Example 1416-Desoxy-6-(3-undecenyloxypheny)-4-piperazin-1-yl-D-galactopyranose

6-Desoxy-6-(3-undecenyloxyphenyl)-4-piperazin-1-yl-(1,2:3,4-diisopropyliden)-D-galactopyranose(101 mg, 166.33 μmol) was treated with 2 n HCl (1.66 ml, 3.33 mmol) andthen heated for 2 h at 80° C. LCMS showed consumption of startingmaterial, the reaction mixture was diluted with H₂O and freeze dried.

Yield: 18 mg (20.5%)

Synthesis of Example 1426-Desoxy-6-(1-(4-(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethoxy)phenyl)piperazin-1yl-D-galactopyranose

The synthesis of example 142 was following the procedure described forexample 141 starting from2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethan-1-ol (500 mg, 2.66 mmol)and 4-methylbenzenesulfonyl chloride (759.63 mg, 3.98 mmol) to yield theactivated spacer (2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl4-methyl-benzenesulfonate, yield: 420 mg, 46.2%).

In parallel6-Desoxy-6-(4-hydroxyphenyl)-piperazin-1-yl-(1,2:3,4-diisopropyliden)-D-galactopyranose(269 mg, 639.71 μmol) was prepared and then coupled with(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl4-methyl-benzenesulfonate (262.85 mg, 767.65 μmol) to yield.6-desoxy-6-(1-(4-(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethoxy)phenyl)piperazin-1yl-(1,2:3,4-diisopropyliden)-D-galactopyranose (180 mg, 47.6%), whichwas then deprotected with aqueous HCl.

Yield: 130 mg (69.5%)

LC/MS (ES-API): m/z=511.38 [M+H]+; calculated: 510.25 tR (λ=220 nm):0.98 (LC/MS—Method 2)

Example 143 Biological Assays 1. HepG2 Assay: Procedure

For measurement of ¹⁴C 2-deoxy-D-glucose transport into HepG2 cells,cells were seeded in collagen treated 96-well plates (Cytostar-T PlatesPerkin Elmer, 40.000 cells/200 μ/well) in medium complete (MEM W/GLUT-I,EARLES (Gibco #41090)/NEAA PS/10% FCS) and grown for 48 hours. Afterstarvation for 2 hours with MEM W/GLUT-I, EARLES (Gibco #41090)/NEAA/noserum 100 μl/well and washing twice with 200 μL KRB buffer, cells werestimulated in a dose dependent manner by adding 20 μL of test compounddilution 0-750 μM (7.5 times higher concentration than final) or 20 μLof 188 μM Cytochalasin B solution as negative control, to 80 μL KRBbuffer and incubated for 30 minutes. After compound stimulation, thetransport of ¹⁴C 2-deoxy-D-glucose was started by adding of 50 μL ¹⁴C2-deoxy-D-glucose solution (250 μM 2-deoxy-D-glucose cold and 15 μM ¹⁴C2-deoxy-D-glucose 0.13 μCi/well) for 20 minutes. Transport was stoppedby adding 50 μL/well 40 μM Cytochalasin B solution. Plates were measuredin a 96-well Wallac Microbeta device. The cpm (counts per minute) valueswere used to determine the % inhibition values for the test compoundswithin each experiment, which then are averaged over the number ofexperiments performed.

Table of results: % inhibition of ¹⁴C-2-deoxy-glucose Example transport[100 μM] SD 1 6.32  6.32 ± 3.81 2 6.50  6.50 ± 4.16 6 7.75  7.75 ± 3.287 6.99  6.99 ± 2.52 8 7.67  7.67 ± 5.12 9 5.11  5.11 ± 3.56 12 5.43 5.43 ± 6.31 14 5.67  5.67 ± 5.11 19 6.89  6.89 ± 6.28 21 7.53  7.53 ±3.59 22 11.40 11.40 ± 3.95 27 13.06 13.06 ± 1.96 28 9.95  9.95 ± 1.83 2918.44 18.44 ± 3.80 34 20.47 20.47 ± 3.07 36 20.63 20.63 ± 3.24 37 6.88 6.88 ± 1.71 38 12.70 12.70 ± 1.51 39 10.99 10.99 ± 4.75 40 10.08 10.08± 7.98 45 29.31 29.31 ± 4.46 46 8.76  8.76 ± 2.67 47 13.82 13.82 ± 5.2348 28.82 28.82 ± 4.82 49 19.91 19.91 ± 3.87 50 13.84 13.84 ± 1.91 5117.86 17.86 ± 6.82 52 14.55 14.55 ± 3.95 53 23.89 23.89 ± 5.32 54 10.4810.48 ± 8.16 55 12.39 12.39 ± 2.89 56 21.08 21.08 ± 2.76 57 24.14 24.14± 5.94 58 19.98 19.98 ± 5.00 59 7.56  7.56 ± 4.50 60 14.75 14.75 ± 4.7261 9.42  9.42 ± 6.75 63 37.62 37.62 ± 4.09 64 8.86  8.86 ± 3.67 65 7.79 7.79 ± 2.92 66 5.12  5.12 ± 6.07 67 25.77 25.77 ± 3.88 69 6.14  6.14 ±6.61 75 31.19 31.19 ± 6.64 79 25.54 25.54 ± 3.49 88 31.37 31.37 ± 1.7592 23.10 23.10 4.60

2. Glucose Displacement Assay (ATP Measurement)

Reagent Provider Catalogue n. CellTiter-Glo ® Luminescence PromegaG-7571 Cell Viability Assay A2780 Human Carcinoma Cell ECACC 93112519line 96-well LIA plate, white Greiner Bio-one 655073 RPMI 1640 mediumGlutaMAX Thermo Fisher 61870 Scientitic RPMI 1640 medium (no glucose)Thermo Fisher 11879 Scientific Fetal Bovine Serum Pan Biotech P-30-3305D-(+)-Glucose solution Sigma Aldrich G-8644 PBS buffer Life Technologies14190 KRB buffer PAN P05-32500* DMSO Sigma D-2650 Rotenone Sigma R-8875*Customer Formulation, sterile filtered: 1.7 mM CaCl₂ × 2H₂O; 1.2 mMKH₂PO₄; 4.8 mM KCl; 1.2 mM MgSO₄ × 7H₂O; 120 mM NaCl; 26 mM NaHCO₃

30.000 A2780 Human Carcinoma Cells are seeded per well in a Greiner96-well plate. Cells are expanded and cultured in RPMI 1640medium+GlutaMAX® with 10% FCS and 11 mM glucose, at 37° C. with 5% CO2.After 44 h, culture media is changed and washed once with PBS tostarvation media consisting of RPMI 1640 medium with 1% FCS withoutglucose for 2 hours. Cells are then washed with KRB buffer, followedincubation for 20 min at 37° C. of the treatment mix consisting of: 60μL KRB buffer/well with 10 μL of compound or DMSO 10×. 10 μl of rotenoneis added to the mix to a final concentration of 0.5 μM. Cell plates areleft for 2 min at room temperature. 20 μL of different glucoseconcentrations are added to the mix—typically 0.1 to 20 mM range -.Cells are incubated for another 15 min at 37° C., before measuring ATPwith the CellTiter-Glo® Assay, under manufacturer's guidance, butwithout the equilibration step at room temperature for 30 min. In brief,100 μl of Cell-Titer-Glo® Reagent is added to the wells containingalready 100 μl of the previous reaction mix. Plates are mixed for 2 minat 800 rpm, followed by incubation at room temperature for 10 min tostabilize the luminescent signal. Luminescence is then recorded with theTekan Ultra Evolution reader.

Example 140

IC₅₀ 67.9 μM

Example 141

IC₅₀ 84.8 μM

Example 138

IC₅₀ 8.37 μM (>100 μM @ 10 mM Gluc)

Example 124

IC₅₀ 33.2 μM (>100 μM @ 10 mM Gluc)

3. Erythrocyte Dialysis Assay

6-NBD Glc(6-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-Deoxyglucose) is asmall fluorescent glucose derivative, which has been shown to bind toGluT-1 in astrocytes (L. F. Barros et al., JOURNAL OF NEUROCHEMISTRY 109(2009) 94-100). Here 6-NBD-Glc or alternatively 2-NBD-Glc was used indialysis based competition experiments.

Freshly isolated heparinized rat blood samples were immideately diluted(1:5) in PBS buffer to generate a stable stock solution. This stocksolution was further diluted (1:1 or 1:3) for the following dialysisexperiments in Rapid Equilibrium Dialysis (RED) devices (ThermoScientific Pierce).

The aforementioned stock solution was further diluted (1:1 or 1:3respectively) and added to the dialysis compartiment of a RED device. Inparallel stock solution was diluted (1:1) with buffer containing 6 NBDGlc (25 μM).

Dialyses was started by addition of 6 NBD Glc (25 μM) in PBS Buffer(k^(on)) or pure PBS buffer (ko^(off)) respectively into the buffercompartiment. Aliquots (20 μl) were taken after defined time intervalsfrom the buffer compartiment and fluorescence was measured in a UV platereader (Thermovarioskan, Thermo).

As a control, free diffusion of 6-NBD-Glc (25 μM) in PBS buffer vs purebuffer was measured.

Data are given in the following table:

Time 1:1 Dilution 1:1 Dilution Diffusion (min) k^(on) k^(off) (Control)0 55 0 0 30 50.6 3.4 3.7 60 45.1 6.5 7.5 90 33 12 9.2 120 34 16.1 10Graphical evaluation of the different slopes gave a K_(D) = 0.68 for6-NBD-Glc. K^(on) = 0.199; K^(off) = 0.136; K_(D) = 0.136/0.199; K_(D) =0.68: 68% free.

FIG. 1: Fluorescence intensity [AU] is plotted vs time [min].

Squares: Decreasing fluorescence intensity measured in buffercompartiment, due to binding to eyrothrocytes.

Triangles: Increasing fluorescence intensity measured in buffercompartiment, due to release from erythrocytes.

K_(D) was calculated from the slopes: K_(D)=0.68 for 6-NBD-Glc.

Glucose dependency was determined following the protocol describedabove, comparing the following solutions: 6 NBD-Glc (100 μM), 6NBD-Glucose (100 μM)+Glucose 20 mM. Data are shown in the next table

Time 6-NBD-Glc NBD-Glc [50 μM] + (min) [50 μM] Glc [10 mM] 0 0 0 15 7.57.2 30 10.6 11.9 60 11.1 17 90 14.9 18.5 120 15.8 20.3 180 16 22.5

Free concentration of 6-NBD-Glc is increased in presence of Glc [10 mM].

FIG. 2: Fluorescence intensity [AU] is plotted vs time [min].

Increase of fluorescence in buffer compartiment in presence (squares) orabsence of glucose (rhombus).

Example 102 was investigated in a competition experiment following thesame protocol using a 6-NBD-Glc solution (100 μM) in comparison to 6NBD-Glucose (100 μM)+Example 102 (50 μM). The results are shown in thefollowing table

Time Example 102 (min) [25 μM] Control 0 0 0 15 4.9 0.9 30 11.1 2.2 6012.5 3.3 90 19.3 3.8 120 25.1 8 180 32.1 10.1

FIG. 3: Fluorescence intensity [AU] is plotted vs time [min].

In presence of the Example 102 a clear increase in the slope (rhombus)is seen in comparison to control (squares) indicating the competitionwith 6-NBD-Glc.

1. A conjugate of formula (I)P-[L₁]_(m)-[A₁]_(o)-[L₂]_(p)-[A₂]_(r)-[L₃]_(q)-S   (I) wherein P is aninsulin or an insulinotropic peptide, L₁, L₂, and L₃ are independentlyof each other a linker having a chain length of 1-20 atoms, A₁ and A₂are independently of each other a 5 to 6 membered monocyclic ring, or a9 to 12 membered bicyclic ring, or two 5 to 6 membered monocyclic and/or9 to 12 membered bicyclic rings connected to each other, wherein eachring is independently a saturated, unsaturated, or aromatic carbocyclicor heterocyclic ring and wherein each ring may carry at least onesubstituent, S is a sugar moiety which binds to the insulin independentglucose transporter GluT1, and wherein the sugar moiety S comprises aterminal pyranose S1 moiety which is attached via position 2, 4, or 6 tothe conjugate of formula (I), m, o, p, r, and q are independently ofeach other 0 or 1, and wherein at least one of r and o is 1, or apharmaceutically acceptable salt or solvate thereof.
 2. The conjugate offormula (I) of claim 1, wherein P is an insulin which is attached via anamino group, particularly via the amino side chain of an insulin B29Lysresidue or via the amino terminus of an insulin B1 Phe residue.
 3. Theconjugate of formula (I) of claim 1 or 2, wherein L₁, L₂, and L₃ areindependently of each other (C₁-C₂₀) alkylene, (C₂-C₂₀) alkenylene, or(C₂-C₂₀) alkynylene, wherein one or more C-atoms may be replaced byheteroatoms or heteroatom moieties, particularly by O, NH, N(C₁₋₄)alkyl, S, SO, SO₂, O—SO₂, O—SO₃, O—PHO₂, or O—PO₃ and/or wherein one ormore C-atoms may be substituted with (C₁₋₄) alkyl, (C₁₋₄) alkyloxy, oxo,carboxyl, halogen, e.g. F, Cl, Br, or I, or a phosphorus-containinggroup.
 4. The conjugate of formula (I) of any one of claims 1-3, whereinL₃ is (C₁-C₆) alkylene, particularly (C₁₋₄) alkylene, wherein one or twoC-atoms may be replaced by heteroatoms or heteroatom moieties,particularly by O, NH, N(C₁₋₄) alkyl, S, SO, SO₂, O—SO₂, O—SO₃, O—PHO₂,or O—PO₃ and/or wherein one or more C-atoms may be substituted with(C₁₋₄) alkyl, (C₁₋₄) alkyloxy, oxo, carboxyl, halogen, e.g. F, Cl, Br,or I, or a phosphorus-containing group.
 5. The conjugate of formula (I)of any one of claims 1-3, wherein L₃ is C═O.
 6. The conjugate of formula(I) of any one of claims 1-3, wherein L₂ is selected from —CO—(CH₂)₃—,—(CH₂)₆—NH—, —(CH₂)₂—CO—(CH₂—CH₂—O)₂—(CH₂)₂—NH— or —CH₂—O—(CH₂—CH₂—O)₃—.7. The conjugate of formula (I) of any one of claims 1-6, wherein A₁ andA₂ are independently of each other a heterocyclic ring, wherein the ringmay carry at least one substituent.
 8. The conjugate of formula (I) ofany one of claims 1-7, wherein A₁ and A₂ are independently of each otherselected from a 5 to 6 membered monocyclic or a 9 to 12 memberedbicyclic ring, wherein the ring is heterocyclic with 1 to 4 ring atomsbeing selected from N, O, and/or S, and wherein the ring may carry atleast one substituent.
 9. The conjugate of formula (I) of any one ofclaims 1-8, wherein A₁ and A₂ are independently of each other a 5 to 6membered monocyclic ring, wherein the ring is a heteroalkyl ring,particularly selected from pyrrolidinyl, pyrazolidinyl, imidazolidinyl,triazolidinyl, piperazinyl, piperidinyl, morpholinyl, wherein the ringmay carry at least one substituent, or a 9 to 12 membered bicyclic ringwherein the ring is a heteroalkyl ring with 1 to 4 ring atoms beingselected from N, O, and/or S, and wherein the ring may carry at leastone substituent.
 10. The conjugate of formula (I) of any one of claims1-9, wherein A₁ and A₂ are independently of each other 1,2,3-triazolyl.11. The conjugate of formula (I) of any one of claims 1-9, wherein A₂ is1,2,3-triazolyl.
 12. The conjugate of formula (I) of any one of claims1-9, wherein A₂ is piperazinyl.
 13. The conjugate of formula (I) of anyone of claims 1-12, wherein r=1 and A₂ is present and o=0 and A₁ isabsent.
 14. The conjugate of formula (I) of any one of claims 1-12,wherein r=1 and A₂ is present and o=1 and A₁ is present.
 15. Theconjugate of formula (I) of any one of claims 1-14, wherein (i) m=1,o=0, p=0, and q=0 or 1, or wherein (ii) m=1, o=1, p=1, and q=0 or
 1. 16.The conjugate of formula (I) of any one of claims 1-15, wherein A₂ ispiperazinyl, L₂ is absent and A₁ is cyclohexanyl.
 17. The conjugate offormula (I) of any one of claims 1-15, wherein A₂ is piperazinyl, L₂ isabsent and A₁ is cyclohexanyl.
 18. The conjugate of formula (I) of anyone of claims 1-15, wherein A₂ is piperazinyl, L₂ is —CH₂— and A, iscyclohexanyl.
 19. The conjugate of formula (I) of any one of claims1-15, wherein A₂ is piperazinyl, L₂ is absent and A, is phenyl.
 20. Theconjugate of formula (I) of any one of claims 1-15, wherein A₂ is1,2,3-triazolyl, L₂ is absent and A₁ is phenyl.
 21. The conjugate offormula (I) of any one of claims 1-15, wherein L₃ is —CO—, A₁ is phenyl,L₂ is —O— and A₁ is phenyl wherein each ring may be unsubstituted orcarry at least one substituent, for example, 1 to 3 substituentsselected from halogen, NO₂, CN, (C₁₋₄) alkyl, (C₁₋₄) alkoxy,(C₁₋₄)alkyl-(C₃₋₇)cycloalkyl, (C₃₋₇) cycloalkyl, OH, benzyl, —O-benzyl,carboxyl, carboxyester, carboxamide, or mono (C₁₋₄) alkyl, or di (C₁₋₄)alkyl carboxamide.
 22. The conjugate of formula (I) of any one of claims1-15, wherein the group -A₂-L₃- is selected from


23. The conjugate of formula (I) of any one of claims 1-22, wherein thesugar moiety S comprises a terminal pyranose moiety S1 having a backbonestructure of Formula (II)

wherein 1, 2, 3, 4, 5, and 6 denote the positions of the C-atoms in thepyranose moiety, wherein

is a single bond and

is a single or a double bond, R1 and R3 are H or a protecting group,which is attached via position 2, 4, or 6 to the conjugate of formula(I).
 24. The conjugate of formula (I) of claim 23, wherein the terminalpyranose moiety S1 is selected from glucose, galactose, 4-deoxyglucose,and 4,5-dehydroglucose derivatives, wherein the terminal pyranose moietyS1 is attached via position 2, 4, or 6 to the conjugate of formula (I)or mannose attached via position
 6. 25. The conjugate of formula (I) ofany one of claims 23-24, wherein the terminal pyranose moiety S1 is ofthe Formula (IIIa) or (IIIb):

wherein R1 is H or a protecting group, R2 is OR8, or NHR8 or anattachment site to the conjugate of formula (I), wherein R8 is H or aprotecting group, R3 is H or a protecting group, R4 is H, OR8, or NHR8or an attachment site to the conjugate of formula (I), wherein R8 is Hor a protecting group, or R1 and R2 and/or R3 and R4 form together withthe pyranose ring atoms to which they are bound a cyclic group, e.g. anacetal, R5 and R6 are H or together with the carbon atom to which theyare bound form a carbonyl group, R7 is OR8, or NHR8 or an attachmentsite to the conjugate of formula (I), wherein R8 is H or a protectinggroup, and wherein one of R2, R4, and R7 is the attachment site to theconjugate of formula (I).
 26. The conjugate of formula (I) of claim 23or 25, wherein R1 and R3 are H.
 27. The conjugate of formula (I) of anyone of claims 23, 25, or 26, wherein R2 is OR8 or an attachment site tothe conjugate of formula (I), R4 is H, OR8, or an attachment site to theconjugate of formula (I), and R7 is OR8 or an attachment site to theconjugate of formula (I), and wherein R8 is H or a protecting group. 28.The conjugate of formula (I) of any one of claims 23-27, whereinposition 6 of the pyranose moiety S1 and particularly R7 is theattachment site to the conjugate of formula (I).
 29. The conjugate offormula (I) of any one of claims 23-28, wherein the pyranose moiety S1is of formula (IVa), (IVb), (IVc), (IVd), or (IVe):

wherein R1, R2, R3, R5, R6, and R7 are defined as in any one of claims25-28 and wherein R4 is H, a protecting group, or an attachment site tothe conjugate of formula (I), or R4a is H, or an attachment site to theconjugate of formula (I).
 30. The conjugate of formula (I) of any one ofclaims 1-29, wherein the sugar moiety S is of Formula (V):—[X₂—S2]_(s)-X₁—S1   (V) wherein X₁ is a bond or O, particularly a bond,X₂ is a bond, NH or O, particularly a bond, S2 is a mono- ordisaccharide moiety, particularly comprising at least one hexose orpentose moiety, more particularly at least one pyranose or furanosemoiety and S1 is a terminal pyranose moiety as defined in any one ofclaims 23-28, and s is 0 or
 1. 31. The conjugate of formula (I) of anyone of claims 1-30, wherein the saccharide moiety S2 is a pyranosemoiety, particularly selected from glucose, galactose, 4-deoxyglucoseand 4,5-dehydroglucose derivatives, or a furanose moiety, particularlyselected from fructose derivatives.
 32. The conjugate of formula (I) ofclaim 30 or 31, wherein the saccharide moiety S2 is of Formula (VIa),(VIb), (Vic), (VId), or (VIe):

wherein R11 is a bond to X₁, R12 is OR8 or NHR8 or an attachment site toX₂, wherein R8 is H or a protecting group, R13 is H or a protectinggroup, R14 is R8 or an attachment site to X₂, wherein R8 is H or aprotecting group, R14a is H or an attachment site to X₂, R15 and R16 areH or together with the carbon atom to which they are bound form acarbonyl group, R17 is OR8 or an attachment site to X₂, wherein R8 is Hor a protecting group, or R11 and R12 and/or R13 and R14 form togetherwith the ring atoms to which they are bound a cyclic group such as anacetal, and wherein one of R12, R14, and R17 is an attachment site toX₂.
 33. The conjugate of formula (I) of any one of claims 1-32, whichhas an affinity of 10-500 nM to the insulin independent glucosetransporter GluT1.
 34. The conjugate of formula (I) of any one of claims1-33 which reversibly binds to the insulin independent glucosetransporter GluT1 dependent from the glucose concentration in thesurrounding medium.
 35. The conjugate of formula (I) of any one ofclaims 1-34, wherein the sugar moiety S comprises a single terminalsaccharide moiety.
 36. The conjugate of formula (I) of any one of claims1-35 for use in medicine, particularly in human medicine.
 37. Theconjugate of formula (I) of any one of claims 1-35 for use in theprevention and/or treatment of disorders associated with, caused byand/or accompanied by a dysregulated glucose metabolism.
 38. Theconjugate of formula (I) of any one of claims 1-35 for use in theprevention and/or treatment of diabetes, particularly of diabetes type 2or of diabetes type
 1. 39. A pharmaceutical composition comprising aconjugate of formula (I) of any one of claims 1-35 as an active agentand pharmaceutically acceptable carrier.
 40. A method of preventingand/or treating a disorder associated with, caused by and/or accompaniedby a dysregulated glucose metabolism, comprising administering aconjugate of formula (I) of any one of claims 1-35 or a composition ofclaim 39 to a subject in need thereof.
 41. A compound of formula (Ia)R—(O═C)-[L₁]_(m)-[A₁]_(o)-[L₂]_(p)-[A₂]_(r)-[L₃]_(q)-S   (Ia) whereinL₁, L₂, L₃, A₁, A₂, S, m, o, p, r, and q are defined as in any one ofclaims 1-35, R is H, halogen, OH, O-alkyl-, an anhydride forming groupor another active ester forming group, like 4-nitrophenylester,succinate or N-hydroxy benzotriazol, or a pharmaceutically acceptablesalt or solvate thereof.
 42. A compound of formula (Ib)[L₁]_(m)-[A₁]_(o)-[L₂]_(p)-[A₂]_(r)-[L₃]_(q)-S   (Ib) wherein L₁, L₂,L₃, A₁, A₂, S, m, o, p, r, and q are defined as in any one of claims1-35, or a pharmaceutically acceptable salt or solvate thereof.